Erm family binding agents and their use in diagnosis and treatment of proliferative conditions

ABSTRACT

The methods and compositions of the invention provide new diagnostic markers for cancers (e.g., ovarian cancer, PPC, etc.) and other proliferative diseases or conditions such as psoriasis and endometriosis. The methods and compositions of the invention further provide new treatments for such proliferative diseases or conditions.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/591,714, entitled “ANTIBODIES TO ERMPROTEIN BLOCK INVASIVENESS OF CANCER CELLS AND IDENTIFY THE PRESENCE ANDLEVEL OF ERM PROTEINS IN PROLIFERATION CONDITIONS,” and filed on Jul.27, 2004. The teachings of the referenced application are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States. Halfof all men and one-third of all women in the US will develop cancerduring their lifetime. Today, millions of people are living with canceror have had cancer. The sooner a cancer is found and treatment begins,the better are the chances for survival.

To illustrate, ovarian cancer is cancer that begins in the cells thatconstitute the ovaries, including surface epithelial cells, germ cells,and the sex cord-stromal cells. The most common type of cancer arisingfrom the ovary is ovarian epithelial cancer (OVCA). A second, lesscommon cancer (˜10% of expected OVCA's) apparently arises from theperitoneum and has a clinical course indistinguishable from OVCA. Theseso-called primary peritoneal cancers (PPC) may arise from the same roottissue that covers the ovary, the peritoneum, explaining theirsimilarity. Cancer cells that metastasize from other organ sites to theovary (secondary lesions, most commonly breast or colon cancers) arealso encountered, but are not generally considered ovarian cancers.

Even in this era of increased interest in the health needs of women,epithelial OVCA stands out as a deadly disease in special need ofattention. According to the American Cancer Society, ovarian canceraccounts for only 4 percent of all cancers among women, but ranks fifthas a cause of their deaths from cancer. Over 16,000 women die each yearfrom OVCA/PPC, making it the leading cause of death from gynecologicmalignancy (Murphy et al., Sci Am. 275(3): 126-32, 1996).

This high rate of late detection of OVCA is caused by the absence ofgood diagnostic tests. Malignant cells can progress for a long timebefore they become symptomatic locally or their metastases causesymptoms. Pelvic examination is not successful in finding primary OVCA.Present markers, such as CA125, are non-specific and show falsepositives. The result of these failures is that OVCA is usually detectedlate, typically at an advanced stage when metastatic disease is the ruleand the outcome is almost uniformly fatal.

In addition, few patients survive metastatic disease because no curativedrug treatment exists for metastatic OVCA. Accordingly, the mortalityrate attributed to OVCA has not changed significantly in the last 50years despite the availability of new treatments (Choudhury et al., IntJ Cancer. 108(1): 71-7, 2004; Hu and Xing, Curr Opin Mol Ther. 5(6):625-30, 2003; Palm et al., J Nucl Med. 44(7): 1148-55, 2003; Iwamoto etal., Int J Gynecol Cancer. 13(1): 28-31; Argiris et al., Clin CancerRes. 10(4): 1409-20, 2004).

Endometrial cancer (ENDOCA) is the most common form of gynecologicalcancer in women. It arises from the endometrial lining and, unlike OVCA,invades lymphatics and blood vessels to metastasize widely. AlthoughENDOCA cells are of a similar level of aggressivity as OVCA, ENDOCA isnot as lethal because it is early on associated with uterine bleedingthat sets off a diagnostic workup that usually exposes the ENDOCA andresults in treatment before metastases occur. However, there is nosimple or painless method of performing this evaluation. Usually, anendometrial biopsy is required, and thus repeated testing is notacceptable. Moreover, endometrial biopsies have a low but definite rateof false negatives.

Thus, both OVCA and ENDOCA are examples of cancers which requiresymptoms to reach a significant threshold for detection by presentmethods of diagnosis. However, by then it is often too late for OVCAtreatment, and usually results in hysterectomy and infertility in thecase of ENDOCA.

Thus there is a need to develop new cancer diagnosis and treatmentmethods, particularly those for diagnosing and/or inhibiting cancerinvasion and/or metasasis, and particularly for cancers such as OVCA andENDOCA.

SUMMARY OF THE INVENTION

The instant invention is partly based on the discovery that expressionof ERM family proteins (such as ezrin expression) is correlated with anumber of indications relating to proliferative diseases, such as cancerand certain benign proliferative diseases. For example, expression ofthe ERM family proteins (such as ezrin) is correlated with cellmotility, invasion, and metastasis. Ezrin is over-expressed inmetastatic cancer to a greater extent than primary cancers, which inturn express higher levels than normal tissues. Ezrin expression levelsalso correlate with invasive behavior and prognosis of certain cancers,such as endometrial and ovarian cancers and cancer cells.

It is important to note that ezrin and the other ERM proteins arepresent at relatively low levels or even absent in most normal tissues.The expression of the ERM proteins that have high biochemical homologyis tissue-dependent. ERM proteins may be co-expressed in a cell/tissue,or they may be solitary. The lower expression in normal cells than inhighly proliferating cells and cancers indicates that ezrin orERM-targeted therapies should be well tolerated, since normal cellsappear to be less dependent on ERM proteins. The significant correlationof specific ERM over-expression to specific cell types, such as cancers,makes the therapeutic index of such treatment likely to be veryfavorable. The correlation between ERM expression and certainproliferative disease conditions also indicates that ERM expression is auseful disease marker.

The instant invention is also partly based on the discovery thatantagonists to ERM family proteins, including extracellular antagonists(e.g., anti-ERM antibodies that are administered in vivo) inhibit thefunction of ERM in cells from ERM-associated proliferative diseases,such as cancer. For example, two different anti-ezrin monoclonalantibodies have been found to be effective in inhibiting invasion ofMatrigel membranes (an accepted index of metastatic phenotype andaction) and cancer cell proliferation by ezrin over-expressing cells,such as OVCA and ENDOCA. Importantly, it is also shown herein that theanti-invasive and anti-proliferative effect of anti-ezrin antiserum isdose-dependent.

The finding that anti-ezrin monoclonal antibodies bind to ezrin in livecells, and inhibit the invasive and proliferative behavior of ezrinover-expressing cancer cells under the same conditions that lead toinhibition of Matrigel membrane penetration and cell proliferation,indicates clinical usefulness of inhibiting ERM protein functions usingERM antagonists (e.g., extracellular ERM antagonists such asantibodies).

The instant invention is also partly based on the discovery that certainfree-floating, ezrin-positive materials are present in culture media andin vivo-collected cell-free biological fluids that have access to theplasma membranes of ERM expressing cells. These findings are supportedby microscopic examinations of such cells and tissues showing thebudding off of processes from ezrin-expressing cells, e.g., OVCA, ENDOCAand normal endometrial cells. These findings indicate that ERM proteinsand/or such free-floating ERM-containing structures, which may bepresent in certain body fluids such as ascetic fluid, endometrialsecretion, blood, urine and endometrial washings from individual womenor men (ERMS are expressed in male cancers such as prostate cancer), canserve as clinical tumor markers. In other words, the discovery of therelationship of ERM proteins to membranes, and the breaking off orshedding of ERM-containing cell surface structures, make certain ERMprotein binding agents useful in detecting and/or measuring the presenceand/or progress of certain proliferative diseases, and/or monitoring theeffects of treatments based on detection of ERMs in bodily fluids.

“Binding agents” for the ERMs include those that bind or interact withat least one ERM family proteins, but does not include antisensepolynucleotides against a polynucleotide encoding the ERM familyproteins.

Thus in one aspect, the invention provides a method of inhibiting aproliferative condition in an individual, comprising administering tothe individual an effective amount of a binding agent which binds an ERMfamily protein.

In a related aspect, the invention provides for the use of an effectiveamount of an ERM binding agent for the formulation of a medicament,particularly for the formulation of a medicament for the treatment of aproliferative disorder such as cancer or other proliferative disordersas described herein.

“Inhibit” as used herein includes completely stalling a biologicalactivity (such as cell proliferation, invasion, or metastasis, etc.),preventing or at least delaying the onset of a biological process,and/or reducing the severity and/or symptoms of a biological condition,etc.

The methods of the invention can be used to treat or prevent manyproliferative diseases or conditions associated with ERM familyproteins. As used herein, “proliferative diseases or conditions” includeconditions with excessive cell proliferation and/or cell numberincrease. For example, the proliferative condition includes cancer, suchas ovarian cancer, endometrial cancer (ENDOCA), endometrialadenocarcinoma (such as uterine endometroid adenocarcinoma or “UEC”),primary peritoneal cancer (PPC), renal adenocarcinoma, brainhemangioblastoma, pancreatic adenocarcinoma, epidermoid carcinoma,osteosarcoma, epithelial cancer, melanoma, squamous skin carcinoma,leukemia, breast cancer, glioblastoma, schwannoma, meningioma, malignantmesothelioma, neurofibromatosis, colon cancer, oral cancer, orrhabdomyosarcoma, or other cancers selected from the group consisting oflung cancer, prostate cancer, pancreatic cancer, leukemia, liver cancer,stomach cancer, uterine cancer, testicular cancer, brain cancer,non-hodgkin's lymphoma, hodgkin's lymphoma, Ewing's sarcoma,osteosarcoma, neuroblastoma, rhabdomyosarcoma, melanoma, and braincancer.

In addition, the cancer may be invasive and/or metastatic. In that case,the binding agent of the invention may inhibit cancer invasion and/ormetastasis.

In certain embodiments, the binding agent of the invention also inhibitscell proliferation.

Alternatively, the methods of the invention may be used to treat otherproliferative conditions, such as certain benign proliferativedisorders. These proliferative conditions may include tuberosclerosis,psoriasis, endometriosis, complex endometrial hyperplasia (cH), atypicalendometrial hyperplasia (aH), polyps (such as colon polyps), orneurofibromatosis.

The methods of the invention may be used to treat any individual,including a human patient or a non-human mammal, such as laboratoryanimals (mouse, rat, hamster, rabbit, and other rodents), farm animals(sheep, goat, horse, pig, cattle, etc.), or pets (cat, dog, etc.).

Various ERM protein binding agents may be used in the instant invention.For example, the binding agent may be an antibody, or a functionalfragment thereof. “Functional fragment” includes a fragment that bindsthe antigen, preferably binds the antigen and has at least onefunctional effect of the full antibody (such as inhibit the function ofthe antigen or binding partner), especially when used in the context ofthe subject treatment method. However, functional fragment may only needto be able to bind its intended target molecule for the variousdiagnosis embodiments of the invention. The antibody may be a polyclonalantibody or a monoclonal antibody. The antibody may be a xenogeneic, anallogeneic, or a syngeneic antibody. The antibody can also be a modifiedantibody selected from the group consisting of: a chimeric antibody, ahumanized antibody, and a fully human antibody. The functional fragmentof an antibody may be F(ab′)2, Fab, Fv, or scFv, one or more CDR's, etc.

In addition, the binding agent can be a small molecule antagonist of theERM proteins, such as those with molecular weights no more than about5000 Da, 4000 Da, 3000 Da, 2000 Da, 1000 Da, 500 Da, 200 Da, or lessthan 100 Da. Such small molecule binding agents may be small peptides,or peptidio-mimetics, or any other organic or inorganic compounds thatcan bind any ERM protein and inhibit ERM protein function (such as theirrole in proliferation and/or invasion, metastasis).

The binding agents of the invention may be specific for only one of theERM family proteins, or may be specific for a subset of all ERM familyproteins, or may be pan-generic to most or all of the ERM familyproteins.

The binding agents of the invention may recognize the full-length ERMprotein, or recognize only a portion/fragment of the ERM protein. Forexample, the binding agent may bind to the N-terminal portion of the ERMprotein, e.g., the portion used for ERM-binding to cell-surfacereceptors, such as EGFR. Binding to the ERM N-terminal domain mayfurther inhibit the interaction between the ERM protein with its normalbinding partners, such as a cell surface receptor, e.g., an EGF familyreceptor (e.g., EGFR or c-erbB2), an IGF family receptor, an EstrogenReceptor, an IL-1a receptor, CD43, or CD44.

One or more of the binding agents of the invention recognizing anon-overlapping or overlapping region of the same or different ERMfamily proteins may be used simultaneously and/or sequentially.

The binding agents of the invention may recognize both the “closed”(“dorman” or inactive version of the ERM protein that is released fromthe protein-synthetic machinery of the cell) and the “open” (oractivated) version of the ERM protein, or only one form but not theother.

One representative member of the ERM family proteins is ezrin. Othermembers of the ERM family proteins include moesin, radixin, or NF2(Neurofibromatosis Factor 2)/merlin/schwannomin (SCH). Other ERM-relatedproteins may include protein 4.1 and talin. These proteins may berepresented in the cell or free-floating ERM-containing structures as“wild type” proteins, or modified proteins, such as those found in OVCAand other cancers, and deposited in the GenBank (see below). These mayalso include NF2, which in cases of neurofibromatosis is a mutated ERMand lacks a portion of the molecule that ceases itsproliferation-regulating function.

The method of the invention may additionally comprise administering asecond therapeutic agent that is effective against the proliferativedisease or condition, sequentially or concurrently with the ERM bindingagent

For example, where the proliferative condition is cancer, the secondtherapeutic agent may be selected from the group consisting of:methotrexate, amsacrine, azacytidine, bleomycin, busulfan, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,cyclophosphamide, cytarabine, dactinomycin, daunombicin, decarbazine,docetaxel, doxorubicin, epirubicin, estramustine, etoposide,floxuridine, fludarabine, fluorouracil, gemcitabine, hexamethylmelamine,idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine,melphalan, mercaptopurine, mitomycin C, mitotane, mitoxantrone,oxaliplatin, paclitaxel, pemetrexed, pentostatin, plicamycin,procarbazine, ralitrexed, semustine, streptozocin, temozolamide,teniposide, thioguanine, thiotepa, topotecan, trimitrexate, valrubicin,vincristine, vinblastine, vindestine, vinorelbine, aminoglutethimide,anastrozole, asparaginase, bcg, bicalutamide, buserelin, campothecin,clodronate, colchicine, cyproterone, dacarbazine, dienestrol,diethylstilbestrol, estradiol, exemestane, filgrastim, fludrocortisone,fluoxymesterone, flutamide, genistein, goserelin, hydroxyurea, imatinib,interferon, ironotecan, letrozole, leucovorin, leuprolide, levamisole,medroxyprogesterone, megestrol, mesna, nilutamide, nocodazole,octreotide, pamidronate, porfimer, raltitrexed, rituximab, suramin,tamoxifen, temozolomide, testosterone, titanocene dichloride,trastuzumab, tretinoin, vindesine, HERCEPTIN® and other antibodytherapeutics, and an anti-sense or RNAi agent against one or more genespromoting the progression of the cancer.

Furthermore, the individual receiving the treatment may be additionallysubjected to radiation therapy and/or surgery, with or withoutcontinuing or re-starting the anti-ERM therapy.

Another aspect of the invention provides a method of diagnosis for, oraiding in the diagnosis of, a proliferative disorder in an individual,comprising determining the amount and/or concentration of an ERM familyprotein in a sample from an individual suspected of having, or at riskof having, the proliferative disorder, wherein an amount and/orconcentration of the ERM family protein that is significantly higherthan a normal or control sample is indicative of the existence of theproliferative condition in the individual. The method may similarly beused to diagnose other disorders associated with over-expression of ERMproteins.

The method may use a sample such as a body fluid from the individual.Many different types of body fluids may be used, including peritonealfluid, ascitic fluid, endometrial secretion, blood, serum, urine, semen,or lymph fluid.

The assay may be done in various ways, including an Enzyme LinkedImmunoSorbant Assay (ELISA), in which a first immobilized binding agent(e.g., immobilized on a solid surface such as a 96-well plate, etc.) isused to bind and isolate an ERM protein in a fluid, and a seconddetection binding agent (such as a binding agent labeled by afluorescent dye, an enzyme, or a radio label) is used to bind the boundERM protein. The presence and amount of the labeled second detectionbinding agent may then be determined/measured.

According to the subject method, the amount and/or concentration of theERM family protein detected in the sample is proportionally indicativeof the severity and/or extent of the proliferative condition.

In certain embodiments, the amount and/or concentration of the ERMfamily protein may be used along with the results of one or more otherdiagnostic tests, such as those selected from the group consisting of:mammography, an early mammography program, a frequent mammographyprogram, a biopsy procedure using a tissue of the individual, anultrasound analysis of a suspected disease organ and optionally a normalorgan, a magnetic resonance imaging (MRI) analysis of a suspecteddisease organ and optionally a normal organ, an electrical impedance(T-scan) analysis of a suspected disease organ and optionally a normalorgan, ductal lavage, a nuclear medicine analysis (e.g.,scintimammography), sequence analysis of one or more disease-associatedgenes (e.g., BRCA1 and/or BRCA2, etc.), and a thermal imaging of asuspected disease organ and optionally a normal organ.

The diagnosis method of the invention can be used for diagnosis of avariety of proliferative diseases/conditions, including cancer. Thecancer may be ovarian cancer, endometrial cancer (ENDOCA), endometrialadenocarcinoma (such as uterine endometroid adenocarcinoma or “UEC”),primary peritoneal cancer (PPC), renal adenocarcinoma, brainhemangioblastoma, pancreatic adenocarcinoma, epidermoid carcinoma,osteosarcoma, epithelial cancer, leukemia, breast cancer, glioblastoma,schwannoma, meningioma, malignant mesothelioma, neurofibromatosis, coloncancer, oral cancer, or rhabdomyosarcoma, etc. In addition, the cancermay be invasive and/or metastatic, or may be benign.

The diagnosis method of the invention can also be used for diagnosis ofbenign proliferative disorders, such as tuberosclerosis, psoriasis,endometriosis, endometrial or other tissue hyperplasia, complexendometrial hyperplasia (cH), atypical endometrial hyperplasia (aH),polyps (such as colon polyps), or neurofibromatosis. Such conditionsinclude paoriasis, or endometriosis.

The diagnosis method of the invention can be used for diagnosis in ahuman or a non-human mammal, such as laboratory animals (mouse, rat,hamster, rabbit, and other rodents), farm animals (sheep, goat, horse,pig, cattle, etc.), or pets (cat, dog, etc.).

The diagnosis method of the invention may be performed, e.g., the amountand/or concentration of the ERM family protein is determined, using abinding agent which binds the ERM family protein. The binding agent maybe an antibody, or a functional fragment thereof. “Functional” may onlyrequire the ability to bind in the context of the subject diagnosismethods. The antibody may be a polyclonal antibody or a monoclonalantibody. The antibody may be a xenogeneic antibody, an allogeneicantibody, or a syngeneic antibody. The antibody may be a modifiedantibody selected from the group consisting of: a chimeric antibody, ahumanized antibody, and a fully human antibody. The functional fragmentmay be F(ab′)2, Fab, Fv, scFv, or one or more CDR's.

In addition, the binding agent may be a small molecule with molecularweight no more than about 5000 Da, 4000 Da, 3000 Da, 2000 Da, 1000 Da,500 Da, 200 Da, or less than 100 Da. Such small molecule binding agentsmay be small peptides, or peptidio-mimetics, or any other organic orinorganic compounds that can bind ERM protein.

In certain embodiments, the binding agent may be immobilized on, forexample, a solid support. For instance, the binding agents may bearranged in a spacially resolved pattern in a binding agent array.

In certain embodiments, the binding agent may also be tagged by a label,such as a fluorescent label, an enzyme label, or a radio-label.

The diagnosis methods of the invention may be used to detect all ERMfamily proteins. A representative ERM family protein is ezrin. Other ERMfamily proteins may include moesin, radixin, or NF2 (NeurofibromatosisFactor 2)/merlin/schwannomin (SCH). Additional ERM-related proteins mayinclude protein 4.1 and talin.

Alternatively, the binding agents of the invention may be specific foronly one of the ERM family proteins, or may be specific for a subset ofall ERM family proteins, or may be pan-generic to most or all of the ERMfamily proteins.

The binding agents of the invention may recognize the full-length ERMprotein, or recognize only a portion/fragment of the ERM protein. Forexample, the binding agent may bind to the N-terminal portion of the ERMprotein, e.g., the portion used for ERM-binding to cell-surfacereceptors, such as EGFR. Binding to the ERM N-terminal domain mayfurther inhibit the interaction between the ERM protein with its normalbinding partners, such as a cell surface receptor, e.g., an EGF familyreceptor (e.g., EGFR or c-erbB2), an IGF family receptor, an EstrogenReceptor, an IL-1α receptor, CD43, or CD44.

One or more of the binding agents of the invention recognizing anon-overlapping or overlapping region of the same or different ERMfamily proteins may be used simultaneously and/or sequentially.

The binding agents of the invention may recognize both the “closed”(“dormant” or inactive version of the ERM protein that is released fromthe protein-synthetic machinery of the cell) and the “open” (oractivated) version of the ERM protein, or only one form but not theother.

Yet another aspect of the invention provides a complex comprising an ERMfamily protein binding agent bound to an extracellular ERM protein,wherein the extracellular ERM protein is on or near the extracellularplasma membrane surface of a cell, such as a cancer cell, or a benignproliferative cell, especially if it is a precancerous lesion. Thecancer cell may be invasive and/or metastatic. In certain embodiments,the ERM protein of the complex may be within a free-floatingERM-containing cell surface structure.

In certain embodiments, the ERM family protein binding agent may belabeled by a moiety, such as a fluorescent dye, an enzyme, or aradio-imaging reagent.

Yet another aspect of the invention provides an in vivo complexcomprising an ERM family protein binding agent bound to an ERM protein.

The in vivo complex may be formed by administering the ERM familyprotein binding agent to an individual having the extracellular ERMprotein. In this embodiment, the individual may be a patient sufferingfrom a cancer or a proliferative disorder.

Alternatively, the in vivo complex may be formed in vitro, and is thenadministered to an individual as a pharmaceutical composition. In thisembodiment, the individual may be healthy, and the complex may conferprophylactic benefits to the individual. The individual may also be apatient suffering from a cancer or a proliferative disorder associatedwith ERM family proteins, or an individual having substantial risk ofsuffering from a cancer or a benign proliferative disorder associatedwith ERM family proteins.

The pharmaceutical composition of the invention may also comprise apharmaceutically acceptable salt, excipient, and/or carrier for in vivoadministration to an individual.

Yet another aspect of the invention relates to a method of treatment orprevention for a proliferative condition in an individual, comprisingadministering to the individual an effective amount of a complexcomprising an ERM family protein binding agent bound to an extracellularERM protein.

In one embodiment, the method reduces or eliminates metastatic spread ofthe cancer.

It should be understood that, to illustrate the claims, Applicants haveconcentrated on the role of one ERM family protein (e.g., ezrin) in tworepresentative cancers (e.g., ovarian cancer and endometrial cancer).However, the compositions and methods of the invention extend further toinclude other normal and abnormal ERM family proteins, and/or otherproliferative disorders, and the effects of classes of antagonists orbinding agents that include antibodies, and functional portions asdiagnostic and/or therapeutic agents.

The embodiments described above, including those described underdifferent aspects of the invention, are contemplated to be applicablefor all aspects of the inventions wherever appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a modified drawing of the cellular location and molecularsites of action of ezrin. Note that the folded, dormant protein thatleaves the Golgi apparatus is moved to the cell membrane and unfoldsthere, placing its N-terminus in the area of the cell membrane. Theactivated ERM is shown binding (by phosphorylation) to thetransmembrane/intracellular domain of the epithelial growth factorreceptor (EGFR). The result is the failure of development of themetastatic phenotype and blocked cell division. For elucidation and as acomparison with a narrower, but successful and theoretically similarapproach, the humanized anti-EGFR anti-cancer immunotherapy drugHERCEPTIN® is depicted as binding to the extra-cellular domain of theEGFR.

FIG. 2 shows the effects of ERM proteins on cell morphology, andillustrates the variety and number of sites that can be attacked by theERM-binding agent, in comparison to the possible number of sitesattacked by HERCEPTIN®.

FIG. 3 shows the result of a Western blot analysis, indicating thatezrin over-expression is related to the stage of OVCA progression. The“*” and “**” represent statistically significant differences.

FIG. 4A shows the result of Matrigel invasion assays, indicating thatanti-ezrin antibody administered to live endometrial cancer cells inculture inhibits invasive behavior of the cancer cells,

FIG. 4B shows that this action occurs in a dose-responsive manner. Notethat in FIG. 4B, the results are presented as % inhibition of Matrigelpenetration.

FIG. 5 illustrates how Matrigel membrane penetration by OVCA cells isinhibited in a dose-dependent manner by anti-ezrin monoclonal antibody.The three data points represent decreasing dilutions of monoclonalantibodies (mAb) at 1:1000, 1:500, and 1:100 dilutions. The verticalbars represent relative numbers of OVCA cells following 24-hourtreatment with anti-ezrin nAb (1:1000 or 1:500 dilutions) or controluntreated cultures.

FIG. 6 shows that anti-ezrin mAb also inhibits OVCA cell growth (e.g.,increase in cell number) by the same starting cells that are in theabove figures. These cells are grown separately, in flasks, to assesscell growth or increase in cell number. The number of cells in theflasks at the end of a 24-hour period are counted. The results fromcells treated with 1:1000 or 1:500 dilutions of anti-ezrin mAb arecompared with that of the control untreated cultures. At the end of theexperiment, the control group had four times as many cells as the 1:500dilution of anti-ezrin mAb (e.g., about 75% inhibition). The decrease incell number in mAb-treated culture may be due to inhibited cellproliferation or increased apoptosis, or both.

FIG. 7 shows that anti-ezrin antibody penetrates a caveolus membrane tobind its target. It is the immunolabeling of ezrin by anti-ezrinantibody (Ab) administered to live OVCA cells in culture. The labelingis performed after the anti-ezrin Ab was added to the OVCA cell culturefor five minutes, after which the cells were washed off antibodies, andthen fixed and labeled by the diaminobenzidine method that labelsantibodies to the anti-ezrin immunoglobulin with electron-densematerial. This is the black matter seen within the cell membranes of theOVCA cells. This has occurred in a typical caveolus, which is where mostof the actions ascribed to ERM proteins occur.

FIG. 8 shows Western analyses of cell-free ascetic fluid from patientswith metastatic OVCA. Note the heavy band that indicates ezrin, which isabout 80 kDa against the molecular sizing markers in the first lane.Note also that there are subsidiary bands that may indicate the presenceof fragments of ezrin and/or variants of ezrin protein produced by thecancer cells. Several such variants have been deposited in the GenBank(see below).

DETAILED DESCRIPTION OF THE INVENTION

Cancer is an abnormal state in which uncontrolled proliferation of oneor more cell populations interferes with normal biological functioning.The proliferative changes are usually accompanied by other changes incellular properties, including reversion to a less differentiated, moredevelopmentally primitive state; the ability to invade surroundingtissues/organs (e.g., invasion); and/or the ability to metastasize todistant tissues/organs (metastasis).

The development of cancer consists of multiple, sequential, andinterrelated steps that lead to the generation of an autonomous clonewith aggressive growth potential. These steps include sustained growthand unlimited self-renewal through a process of autonomous growthsignals, decreased sensitivity to growth-suppressive signals, andresistance to apoptosis. Genetic or cytogenetic events that initiateaberrant growth sustain cells in a prolonged “ready” state by preventingapoptosis.

In addition to the loss of regulated growth, another feature of manytumors is invasion of surrounding tissues. Local invasive infiltration,as in OVCA, and escape to implant in distant sites, as in ENDOCA, arekey and deadly features in many tumors. These actions are accompanied byremodeling of the local vasculature and destruction of surroundingnormal tissues. In addition to the ability of malignant cells to formthin, clawing processes (ruffles and protrusions) and invade physicallybetween cells, the invasive character of certain tumors appears todepend partly on remodeling of the surrounding extracellular matrix,such as by the proteolytic destruction of the extracellular matrixcomponents. One class of enzymes that is upregulated in certain tumorsare the matrix metalloproteinases (MMPs), zinc dependent proteolyticenzymes, that cleave extracellular matrix (collagen, laminin,fibronectin, etc) as well as non-matrix substrates (growth factors, cellsurface receptors, etc). The deregulation of MMPs is involved in manydiseases, such as tumor metastasis, rheumatoid arthritis, andperiodontal disease.

The ERM family of proteins are well-known as intracellularmembrane-actin cross-linking proteins. Ezrin (also known as Cytovillinor Villin-2) is a prototypical member of the ERM protein family. Likeits family members, ezrin is an important signal transduction proteinthat undergoes phosphorylation and translocation on stimulation bygrowth factors and other agents, such as estrogen. The chief partners inthese actions are the contractile protein actin, that forms thecytoskeletal basis for the normal cell specialization and metastaticcell phenotype that are induced by ERM proteins, and proteins that areassociated with the cell membrane, such as cell surface receptors, ERFR,IGF1R, etc. By linking these two types of proteins, the ERM proteins areindispensable to a variety of cellular functions, such as cell surfacespecialization, cell division, adhesion, migration, and the organizationand function of cell surface structures, as described herein in regardto cancer cells.

In the context of cancer and other benign proliferative conditions, ERMproteins play roles in all steps of cancer progression (e.g., sustainedgrowth, unlimited self-renewal, decreased sensitivity togrowth-suppressive signals, and resistance to apoptosis, etc.).Overexpression of ERM proteins is found in many different cancers andbenign proliferative conditions. One of the effects of ERM proteins incancer and benign proliferative conditions may be to foster celldivision, in part by the manipulation of the actin cytoskeleton. Theover-expression of the ERM proteins needed for this action alsounderlies the potential usefulness of ERM proteins as tumor markers. Inaddition, Applicants have shown that development of ruffles andprotrusions is accompanied by MMP2 expression in OVCA cells that areincreasing their invasive activities.

The ERM family of proteins are linked to the intracellularmembrane-actin via phosphorylation at the “C” and “N” termini of theactivated ERM molecule. The C terminus links to actin or forms polymerswith other ERM proteins; while the N-terminus is closest to themembrane, and it links to the transmembrane or cytoplasmic domain ofother proteins via specific tyrosine phosphorylation. The latter linkagemay require binding to spacer molecules known as ezrin/moesin-bindingprotein 50 (EBP50, also known as the sodium-hydrogen transportermolecule). Since they are among the most common transmembrane receptormolecules, growth factor ligation and signal transduction that triggersauto-phosphorylation of the receptor is among the most common sequenceof events leading to binding and action of the ERM proteins. The exampleof EGFR ligation and its subsequent binding to ezrin is mentionedherein, because of the clinical relevance of the success of thehumanized antibody to EGFR HERCEPTIN® that depends on indirect blockadeof the ERM signal transduction pathway for its anti cancer action. TheERM proteins are involved in a variety of cellular functions, such ascell adhesion, migration, and the organization of cell surface thatconcern specialization such as brush borders, dense junctions, andmembrane specializations necessary for invasive behavior by normal andmalignant cells. Since proliferating cells must divide, the ERM proteinsare necessary for cell division and proliferation. The failure of celldivision is often followed by cell death (apoptosis). The loss of theC-terminus of NF2 results in cell proliferation without apoptosis,leading to neurofibromatosis. Applicants have shown the hierarchicalexpression of ERM proteins in cells that are undergoing proliferation,such that more ERM proteins are produced in more rapidly growing cells,such as cancer cells, proliferative phase endometrial gland cells andothers. While not wishing to be bound by any particular theory, thesecells have fragile specialized structures (such as microvilli andprotrusions) that have the ERM proteins in high concentration, fragmentsof these structures are constantly breaking off into the intercellularspace or to body cavities, from which they access the circulation or thebody fluids (such as peritoneal fluids, ascitic fluids, endometrialsecretions, blood, semen, or urine, etc.).

Therefore, the instant invention is partially based on the discoverythat ERM family proteins are markers for proliferative disorders. Thisis based on the over-expression of ERM family proteins by cancers(including OVCA, ENDOCA, and PPC) and benign proliferative conditions.Fragmentation of the ERM-laden cell specializations (protrusions,ruffles, microvilli, etc.) releases ERM's into tissues or body cavities,and thereby into various biological fluids. For example, the presence ofthe ERM's is indicated by the presence of ezrin in cell-free asciticfluid from OVCA patients. Fragments of cells with densely bound ERMproteins can furnish ERM proteins as marker proteins for diagnosis andevaluation of treatment effectiveness. Thus quantitative and qualitativetests and measurement of one or more ERM proteins, including alteredERM's found in normal cells and/or cancers, constitute valuablediagnosis/prognosis/monitoring assays for ERM-associated conditions.

The instant invention thus provides a method to use ERM binding agentsas detection agents for detecting and/or quantitating the ERM proteinsin a number of pathological conditions, using samples such as bodyfluids (e.g. peritoneal fluid, ascitic fluid, endometrial secretion,blood, serum, urine, semen, lymph fluid, etc.) obtained from anindividual suffering from such conditions, or at risk of developing suchconditions. One of the main advantages of the subject method isspecifically due to the lack of ezrin expression by normal tissues. Forexample, normal ovary only expresses ezrin in the depths of the ovarianclefts and in areas where ovarian adhesions have formed. Therefore,sensitivity and specificity of the subject method are high, while falsepositive rate is low.

As a result, the subject method can detect a low level of true positivesignal, thus providing a method for early detection and diagnosis ofdiseases where early diagnosis is critical for prognosis.

The subject diagnosis method not only provides an earlydiagnosis/screening means for certain proliferative diseases, but alsoprovide a non-invasive means to monitor the progress of the disease overtime, its responsiveness to various treatments, and/or the possiblerecurrence of diseases previously in remission. Thus the term“diagnosis” includes not only the initial diagnosis, but also themonitoring of disease progress, the response of the disease to specifictreatment regimens, the detection of possible recurrence, and screeninghealthy individuals or individuals at high risk of developing thesubject disease conditions, etc.

The instant invention is also partially based on the discovery that ERMbinding agents (e.g., antibodies) can effectively be used in treatmentof cancers, pre-cancers, and proliferative disease and like conditions.This is based on at least two findings. First, Applicants have shownthat anti-ezrin antibodies administered to live cells bind ezrin. Thisis substantiated by morphological proof. Second, Applicants observedthat, following the administration of (two different) anti-ezrinantibodies, there is a dose-dependent inhibition of two biologicalactions, e.g., Matrigel membrane penetration and cell growth (cellnumber increase). Thus Applicants have unequivocally show that ERMbinding agents, such as antibodies, can inhibit the actions of ezrin.

These circumstances for the first time demonstrate that ERM proteins canbe self-anchored within the plasma membrane, and that such ERM proteinsare accessible to ERM family protein binding agents, such as anti-ERMantibodies. Most importantly, Applicants demonstrate for the first timethat ERM binding agents, such as ERM antibodies administered to livecells effectively inhibit ERM-associated invasion and/or metastaticbehavior. Thus the invention provides a method to treat or prevent anumber of proliferative conditions, such as cancer or otherproliferative conditions, comprising administering to an individual inneed of such treatment an effective amount of an ERM binding agent astherapeutic agents.

Various aspects of the instant invention are described in more detailbelow.

Exemplary ERM Proteins

Ezrin also known as Cytovillin or Villin 2, is known as a microvillarcytoplasmic peripheral membrane protein that is expressed strongly inplacental syncytio-trophoblasts and in certain human tumors. It is alsoa component of the microvilli of intestinal epithelial cells that servesas a major cytoplasmic substrate for certain protein-tyrosine kinases.

The so-called “ERM proteins,” ezrin, radixin, and moesin, act as linkersbetween the plasma membrane and the actin cytoskeleton. They areinvolved in a variety of cellular functions, such as cell adhesion,migration, and the organization of cell surface structures. They arehighly homologous, both in protein structure and in functional activity,with merlin/schwannomin, the NF2 tumor suppressor protein. The genomicstructures of ezrin and moesin are highly conserved, suggesting thatthey diverged recently (Majander-Nordenswan et al., Hum. Genet. 103:662-665, 1998).

Ezrin is a highly charged protein with an overall pI of 6.1 and acalculated molecular weight of about 69,000, and runs on a gel at about80 kDa as compared to a molecular weight size standard. Highest ezrinexpression was found in intestine, kidney, and lung. The ezrin cDNAclone hybridized to DNAs from widely divergent organisms, indicatingthat the sequence is highly conserved throughout evolution. Within itsN-terminal domain, ezrin also showed a high degree of similarity ofamino acid sequence to the erythrocyte cytoskeletal protein band 4.1.

Moesin stands for membrane-organizing extension spike protein (Lankes etal., Biochem. J. 251: 831-842, 1988). It was first isolated from bovineuterus, and further studies indicated that it shares a significantsequence homology to ezrin, protein 4.1, talin, radixin, and merlin.These proteins constitute a family with structural and probablyfunctional relationships; all of them are localized to the submembranouscytoskeleton. Moesin is widely expressed in different tissues in cells,where it is localized to filopodia and other membranous protrusions thatare important for cell-cell recognition and signaling and for cellmovement.

Lankes and Furthmayr (Proc. Nat. Acad. Sci. 88: 8297-8301, 1991) clonedand sequenced the complete cDNA of moesin, which contains no apparentsignal peptide or transmembrane domain.

Radixin functions as a membrane-cytoskeletal crosslinkers in actin-richcell surface structures and is thereby thought to be essential forcortical cytoskeleton organization, cell motility, adhesion andproliferation. Cloning of the murine and porcine radixin cDNAsdemonstrated a protein highly homologous to ezrin and moesin. Wilgenbuset al. (Genomics 16: 199-206, 1993) cloned and sequenced the humanradixin cDNA and found the predicted amino acid sequence for the humanprotein to be nearly identical to those predicted for radixin in murineand porcine, indicating that this family of proteins are highlyconserved across species.

Radixin is a modular polypeptide consists of a long, central helix,termed the alpha-domain, which connects an N-terminal4.1/ezrin/radixin/moesin (FERM) domain required for membrane binding anda C-terminal region that contains a major actin-binding motif.Conformational regulation of radixin protein function occurs byassociation of the FERM and C-terminal domains, whereby the membrane-and actin-binding activities are mutually suppressed and the protein isthought to take an inactive “closed” form (Hoeflich and Ikura, Int JBiochem Cell Biol. 36(11): 2131-6, 2004).

Myosin regulatory light chain interacting protein (MIR) also belongs tothe ezrin, radixin, moesin (ERM) family of proteins (Bonhauser et al.,FEBS Lett. 553(1-2): 195-9, 2003).

The ERM family proteins includes mammalian and non-mammalian homologs.The human ezrin protein sequence is available in the NCBI database asNP_(—)003370 (nucleic acid sequence NM_(—)003379.3). The human moesinprotein sequence is available in the NCBI database as NP_(—)002435(nucleic acid sequence NM_(—)002444.2). The human radixin proteinsequence is available in the NCBI database as NP_(—)002897 (nucleic acidsequence NM_(—)002906.3). The human NF2 protein sequence is available inthe NCBI database as NP_(—)000259 (isoform 1), NP_(—)861546 (isoform 2),NP_(—)861964 (isoform 3), and NP_(—)861965 (isoform 4). Theircorresponding nucleic acid sequences are NM_(—)000268.2, NM_(—)181825.1,NM_(—)181826.1, NM_(—)181827.1, respectively.

In addition, the ERM family proteins also include differentpost-translationally modified forms, such as phosphorylated forms.

Furthermore, the ERM family proteins include various mutant forms found,e.g., in diseased cells, such as cancers. For example, Applicants haveidentified several mutant forms of ezrin in cancers, the sequences ofwhich are deposited in GenBank as AF199015 (partial human ezrin genesequence in human epidermal carcinoma), AF190059 (mutation of ezrin genein glioblastoma), AF189213 (a human ezrin gene mutation in cancer),AF188897 (human ezrin gene mutation in ovarian cancer), and AF188896(mutation of human ezrin gene in brain cancer).

Other variants, homologs, isoforms, fragments, polymorphisms of theabove-described proteins can be readily obtained from a sequencehomology search (such as NCBI BLAST search) in public (such as GenBank,EMBL, etc.) and/or private databases. These sequences may be used toproduce recombinant ERM proteins, and further used in generating ERMprotein antagonists, such as antibodies (e.g., antibodies raised againstthe N-terminal portions of the ERM proteins).

Exemplary ERM-Associated Diseases

The ERM family proteins have been associated with a number of diseaseconditions, all of which are contemplated to be treated/diagnosed by thesubject methods. Some exemplary disease conditions are briefly describedbelow.

Ezrin expression is correlated with cell motility, invasion, and cancermetastasis. It is known that ezrin is over-expressed in metastaticcancer to a greater extent than primary cancers, which in turn expresshigher levels than normal tissues. Ezrin levels correlate with invasivebehavior and prognosis of endometrial and ovarian cancers and cancercells. It is important that ezrin is present at low levels or absent inmost normal tissues which indicates that ezrin-targeted therapies shouldbe well tolerated.

Endometrial adenocarcinoma are the most common gynecologic cancers, andtheir incidence in countries like Japan has increased year by year dueto the ongoing changes in life style (Japan Vital Statistics, Statisticsand Information Department, Ministry of Health and Welfare, Tokyo,1995). UEC (uterine endometroid adenocarcinoma), one of the majorhistologic types of endometrial adenocarcinoma, is thought to progressthrough a series of histologic changes from normal to hyperplasia toadenocarcinoma with the accumulation of genetic alterations in responseto unopposed estrogen stimulation (Key, Mutat. Res. 333: 59-67, 1995).

Applicants have demonstrated that ezrin transcription is required for invitro invasion and is involved in the acquisition of metastaticpotential in endometrial cancer cells (Ohtani et al., Cancer Letters147: 31-38, 1999). Applicants also examined ezrin protein expression in20 cancerous and 33 non-cancerous tissues using immunohistochemistry andWestern blot analysis (Ohtani et al., Cancer Letters 179: 79-86, 2002).The specimens included 20 uterine endometrioid adenocarcinomas (UEC),seven simple endometrial hyperplasias (sH), seven complex endometrialhyperplasias (cH), seven atypical endometrial hyperplasias (aH), and 12samples of normal endometrium (NE). Tissues of primary (P) andmetastatic (M) lesions of endometrial cancers were obtained from fivepatients. Ezrin was specifically expressed in UEC and its precursorlesions. Ezrin expression was significantly higher in aH (P<0.05) andUEC (P<0.001) compared with NE, sH, and cH. In addition, ezrin wassignificantly highly expressed in M compared with P (P<0.05). Ezrinexpression was associated with neither clinical stage norhistopathologic grade of UEC. In immunohistochemistry, ezrin waslocalized in the membrane of metastasized cancer cells, although ezrinwas mainly distributed in the cytoplasm of most cancer cells and someendometrial hyperplastic cells. On Western blot analysis, ezrin was alsodetected in both cytosolic and membrane fractions in aH and UEC, whereasezrin was detected in only cytosolic fraction in sH and cH.

These data are surprising, since, given the widely-believed subcellularlocalization of ERM proteins (i.e., inside the plasma membrane), itwould not have been expected that an ERM binding agent would bind, orinhibit the function of, the ERM proteins. Nor would one expect todetect ERM proteins with such binding agents in the various body fluids.

These data suggest that Ezrin was expressed at significantly higherlevels in UEC than in NE. The specific expression of ezrin indicatesthat ezrin plays an active role in the development of UEC. Endometrialhyperplasias are premalignant precursors of invasive UEC. Ezrin proteinexpression was observed in such precursor lesions and significantlyincreased in aH which progresses to invasive cancer more frequently thansH and cH. Thus, ezrin protein expression may occur relatively early inendometrial tumorigenesis.

In comparison with the primary lesions, cancer cells in invasive andmetastatic lesions showed stronger expression of ezrin protein. Thelevels of ezrin protein expression were higher in the metastatic lesionsthan in the matched primary lesions. The high-metastatic endometrialcancer cells, which revealed high ezrin expression at apost-transcriptional level, showed higher invasive ability and moreaggressive behavior compared with the clonally maternal low-metastaticendometrial cancer cells. Thus, high expression of ezrin protein in themetastatic lesions and cancer cells with high metastatic potential isconsistent with the notion that ezrin is involved in the late process oftumor progression, including invasion and metastasis.

Applicants also conducted ezrin subcellular localization studies, anddemonstrated that translocation of ezrin from the cytoplasm to the areasof the membrane may occur during tumor progression and be associatedwith the metastatic potential. Recent evidence has demonstrated thatfull-length ezrin exists in a dormant state (“closed” or inactive form)in which biologically relevant binding sites are conformationally maskedwith an intramolecular interaction between the amino- andcarboxy-terminal domains (Bretscher, Curr. Opin. Cell Biol. 11:109-116,1999). Some signals such as phosphorylation by growth factors maydisrupt this intramolecular association, allowing the conformationalactivation (the “open” or activated form) and formation of oligomericsurface linking structures (Bretscher, Curr. Opin. Cell Biol.11:109-116, 1999).

Exemplary Antagonists or Binding Agents

As used herein, the binding agents of the present invention include anycompound (agent) which binds to one or more target ERM family proteins.In some embodiments, the compound inhibits the function of the ERMproteins in proliferation, invasion, and/or metastasis. Generally, suchbinding agents act as antagonists of the ERM protein function, and canbe used as therapeutic agents for treatment of a normal or pathologicalcondition associated with the target ERM protein, or diagnostic agentsfor detecting the presence and/or measuring the quantity of ERM proteinsor ERM protein-containing complexes. Such binding agents may include,but are not limited to, a protein, a peptide, a small molecule (e.g.organic molecule), a peptidomimetic, an antibody (e.g., full-length, orfunctional fragment, derivative thereof). In one embodiment, the bindingagents of the invention bind to the N-terminal part of the ERM proteins.For example, in the case of ERM family proteins, one embodiment of theERM binding agents of the invention bind to the ERM domain/motif usedfor cell surface receptor interaction, and prevent the binding of ERMproteins to these cell surface receptors.

“Bind” or its various grammatical variants is used interchangeably with“interact.” It includes specific binding to a given target, such as aspecific ERM family protein (e.g., ezrin). It also includes relativebroad spectrum of binding to several related ERM family proteins,especially when the these proteins bound by the binding agent share highsequence homology (e.g., at least about 50% amino acid sequenceidentity, or at least about 60%, 70%, 80%, 90%, 95%, 97%, 99% or moreidentical), at least high sequence homology in the region bound by thebinding agent.

“Functional” when used in the context such as “functionalfragment/derivative/fusion” of a binding agent includes those fragments(e.g., less than full-length or the complete parent molecule), orderivatives, or fusions with other moieties, that substantially retainthe ability to bind a target molecule bound by their parent molecules.For example, a functional fragment of an antibody, such as Fv, retainsthe V region of the antibody molecule, which can bind an antigen insubstantially the same manner as the complete antibody does. Thefunctional fragment/derivative/fusion of a binding agent may retain atleast about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the parentmolecule binding ability. In certain embodiments, the functionalfragment/derivative/fusion of a binding agent may even possess a higherbinding affinity to the target than the parent molecule does.Preferably, the binding agent or functional fragment/derivative/fusionthereof may inhibit at least one function of the binding partner.

There is a powerful theoretical similarity between the effect ofanti-EGFR antibody (HERCEPTIN®) and the binding agents of the presentinvention. However, the binding agents or antagonists of the inventionconfer several advantages over the traditional antagonists targeting theextracellular domains of a target molecule. For example, one advantageof the ERM binding agents of the invention is that they act moreproximally to signal transduction via the Rho-pathway. Many cell surfacereceptors (such as the CD44, EGF family (EGFR and c-erbB-2, etc.),Estrogen Receptor (ER), IL-1α receptor, and IGF family receptors), uponactivation, lead to ERM protein (e.g., Ezrin) phosphorylation, such asvia the Rho GTPase signaling pathway. ERM-protein phosphorylation leadsto the conversion of the “closed” or dormant forms of the ERM proteinsto their “open” or active forms, allowing the N-terminal part of the ERMproteins to bind the cell surface receptors, while the C-terminal partof the proteins to bind actins. Therefore, binding of the subject ERMbinding agents to the N-terminal part of the activated ERM proteins(which is intercalated in the cell membrane and available to ERM bindingagents administered in vivo) is not dependent on antagonizing a single,specific cell membrane receptor (such as antagonizing EGFR byHerceptin). Rather, such ERM binding agents inhibit the function of manydifferent cancer cell membrane receptors, such as CD44, the IGF family,the EGF family receptors, Estrogen Receptor (ER), IL-1α receptor, etc.This in turn results in a greater therapeutic efficacy.

Another advantage of the ERM binding agents of the invention lies in thefact that ERM proteins (e.g., ezrin) are expressed in amounts severalfolds greater in cancers than in normal tissues. Therefore, it ispossible to give stronger doses of the subject ERM binding agentswithout causing serious side effects (if any). The therapeutic index ofthe instant methods are relatively high.

In certain aspects, the ERM binding agents include a polypeptide whichis a mutated form, a mimic or a fragment of a polypeptide that naturallybinds to the ERM protein. Such polypeptides can bind to the target ERMprotein and, in some embodiments, inhibit its function in, for instance,proliferation, invasion, and/or metastasis. For example, the subject ERMbinding agent may include a soluble polypeptide having the amino acidsequence of EBP50 (such as NHERF and NHERF2), SAP97, palladin, L1, MRP2,L-selectin, Neutral endopeptidase 24.11 (NEP), ICAM-2, ICAM-3, RhoGDI,Db1, CD44, CD43, or the ERM binding portion thereof. Since the ERMbinding region of these full-length proteins is either known or can bereadily determined using art-recognized techniques (such as in vitrobinding assay using various deletion fragments of the protein, etc.),such dominant negative mutated form, mimic or fragments can be readilymade by the skilled person. Other variant binding sequences orpeptidomimetics designed based on these peptides may also be readilyobtained by using art-recognized methods, such as random mutagenesiscoupled with screening for ERM binding.

In certain aspects, the binding agents of the invention can beantibodies, such as antibodies that are specifically reactive with atleast one ERM family proteins. Antibodies may be polyclonal ormonoclonal; intact or truncated, e.g., F(ab′)2, Fab, Fv; xenogeneic,allogeneic, syngeneic, or modified forms thereof, such as humanized orchimeric antibodies. Alternatively, these antibodies may be encoded bypolynucleotides, and expressed upon transfection of such polynucleotidesinto the target cancer cell.

Although monoclonal antibodies (mAbs) generated from hybridomatechnology have proved to be immensely useful scientific research anddiagnostic tools, they have had a limited success in human therapy.Although murine antibodies have exquisite specificity for therapeutictargets, they do not always trigger the appropriate human effector'ssystems of complement and Fc receptors. More importantly, the majorlimitation in the clinical use of rodent monoclonal antibodies is anantiglobulin response during therapy. See Miller et al., Blood 62:988-995, 1983; and Schroff et al., Cancer Res. 54: 879-885, 1985. Thepatient's immune system normally cuts short the therapeutic window, asmurine antibodies are recognized by a human anti-mouse antibody immuneresponse (HAMA). Similar to serum therapy where antisera used toneutralize pathogen in acute diseases and also prophylactically leads to“serum sickness”, the patient treated with rodent mAbs in multiple dosesinvariably raises an immune response to the mAbs, manifesting similarsymptoms to serum sickness. This response can occur within two weeks ofthe initiation of treatment and precludes long-term therapy. Thusefforts have been made to raise human mAbs against therapeutic targetsthrough immortalization of human antibody-producing cells.

To produce therapeutic antibodies with high binding affinity, reducedimmunogenicity (HAMA response), increased half-life in the human bodyand adequate recruitment of effectors functions (i.e., the ability tosummon the body's own natural defense), the techniques of monoclonalantibody production and recombinant DNA technology are combined toovercome the problem associated with rodent monoclonal antibodies.Besides direct generation of fully human antibody, another popularapproach is to humanize rodent monoclonal antibody. See, for example,Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989), andU.S. Pat. No. 5,693,762.

US20040067532A1 (incorporated herein by reference) describescompositions, methods, and kits for efficiently generating and screeninghumanized antibody with high affinity against a specific antigen.According to that method, a library of humanized antibody is generatedby mutagenizing a chimeric antibody template that combines humanantibody framework and antigen binding sites of a non-human antibody.Alternatively, the library of humanized antibody is generated bygrafting essential antigen-recognition segment(s) of the non-humanantibody into the corresponding position(s) of each member of a humanantibody library. This library of humanized antibody is then screenedfor high affinity binding toward a specific antigen in vivo in organismsuch as yeast or in vitro using techniques such as ribosome display ormRNA display. The specific antigen used in the screening can be the oneagainst which the non-human antibody is originally elicited, or anantigen with similar structural features or biological function. Inaddition, the library of humanized antibody may be used in screening forhigh affinity antibody against an antigen that is structurally and/orfunctionally different from the antigen against which the non-humanantibody is originally elicited. These selection processes can beperformed to select antibody having higher affinity in antigen bindingbut lower immunogenecity than rodent monoclonal antibody. The overallprocess can be efficiently performed in a high throughput and automatedmanner, thus mimicking the natural process of antibody affinitymaturation.

While not wishing to be bound by any particular theory, these antibodiesor fragments thereof may bind the ERM proteins on the surface of thecells (such as the intracellular surface of the cell), or ERM proteinsaccessible by extracellular binding agents, and antagonize ERM proteinfunction in proliferation, invasion, and/or metastasis. Alternatively,since ERM proteins are normally thought to be intracellular proteins notaccessible to large extracellular molecules, immune system of the hostmay recognize and eliminate such “foreign” antibody-engaged ERMstructures on proliferative cells, through, for example, natural killercells (NK cells). Thus by providing a subject ERM protein binding agentcomplex (e.g., antibody-engaged ERM complex), the host immune system maybe stimulated or immunized against such ERM-associated proliferativeconditions.

For example, by using immunogens derived from a target ERM protein, orfrom several ERM proteins, anti-protein/anti-peptide antisera ormonoclonal antibodies can be made by standard protocols (see, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal, such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide (e.g., apolypeptide or an antigenic fragment which is capable of eliciting anantibody response, or a fusion protein). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. A full-length or animmunogenic portion of an ERM protein can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies.

Following immunization of an animal with an antigenic preparation of anERM protein, antisera can be obtained and, if desired, polyclonalantibodies can be isolated from the serum. In certain embodiments,polyclonal antibodies (antisera, affinity purified polyclonalantibodies, etc.) may be preferred, since relatively little is knownabout the metabolism of ERM proteins such as ezrin, and thus the use ofpolyclonal antibodies may in some cases detect certain fragments of ERMproteins that may not be bound by the usual monoclonal antibodies.

To produce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, Nature 256: 495-497, 1975), the human B cellhybridoma technique (Kozbar et al., Immunology Today 4: 72, 1983), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp.77-96, 1985). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ERM protein, andmonoclonal antibodies isolated from a culture comprising such hybridomacells.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with an target protein.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Anantibody of the present invention is further intended to includebispecific, single-chain, and chimeric and humanized molecules havingaffinity for a target protein conferred by at least one CDR region ofthe antibody. Techniques for the production of single chain antibodies(U.S. Pat. No. 4,946,778) can also be adapted to produce single chainantibodies. Also, transgenic mice or other organisms including othermammalian species, may be used to express humanized antibodies. Incertain embodiments, such as in the diagnosis methods, the antibodiesmay further comprise a label attached thereto, and are thus able to bedetected (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

In certain specific embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to a targetprotein may comprise administering to a mouse an amount of animmunogenic composition comprising the target protein effective tostimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monoclonal antibody that binds specifically tothe target protein. Once obtained, a hybridoma can be propagated in acell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the target protein. The monoclonal antibody may bepurified from the cell culture.

Such techniques can be used to generate antibodies using wild-type oraltered ERM proteins, such as those encoded by the polynucleotidesequences of GenBank Accession Nos. AF199015, AF190059, AF189213,AF188897, and AF188896.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, an antibody to be used for certaintherapeutic purposes will preferably be able to target an antigen on aparticular cell type, as opposed to antigen in solution. Accordingly, toobtain antibodies of this type, it may be desirable to screen forantibodies that bind to cells that express the antigen of interest(e.g., by fluorescence activated cell sorting), or at least confirm thatthe antibody can bind to ERM proteins (especially on cell surface). Avariety of different techniques are available for testingantibody:antigen interactions to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g. the BIAcore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g. the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), Western blots,immunoprecipitation assays and immunohistochemistry.

In certain aspects, the binding agents of the present invention includea small molecule or a peptidomimetic. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules (e.g., a peptidomimetic). As used herein, the term“peptidomimetic” includes chemically modified peptides and peptide-likemolecules that contain non-naturally occurring amino acids, peptoids,and the like. Peptidomimetics provide various advantages over a peptide,including enhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B 35: 2331, 1979).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29: 251, 1989). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of CCL21 or a chemokine receptor.

As described herein, small molecule compounds may encompass numerouschemical classes, although typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 50, and less than about 5,000 Da, or less than 4,000 Da, or lessthan 3,000 Da, or less than 2,500 daltons. Candidate agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl, sulfhydryl or carboxyl group. Candidate smallmolecule compounds can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds can be modified throughconventional chemical, physical, and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification, andamidification, to produce structural analogs.

The present invention also contemplates anti-tumor therapeutic agentsobtainable from the screening methods described below.

Binding Agent Screening Assays

There are numerous approaches to screening for additional subjectbinding agents. For example, high-throughput screening of compounds ormolecules can be carried out to first identify agents or drugs that bindthe target protein (e.g., an ERM protein). New binding agents (e.g.,antibodies) against a particular domain of a target protein (such as oneor more specific regions of the N-terminal domain of the ERM proteinsused to interact with cell surface receptors) may be raised. Such targetproteins may be normal or wild-type, or can contain variouspost-translational modifications such as phosphorylation, or can bemutated or truncated versions found in disease cells (e.g., cancercells). The binding agents of the invention may specifically recognizeone of these forms, but not other forms; or could recognize a commonepitope present in all these forms, depending on specific needs. Allsuch binding agents may be obtained using the screening methods of theinvention.

Thus the invention also provides methods to screen for binding agentsthat specifically or generically bind to one or more target ERM orperimembrane proteins, and binding agents identified using the subjectscreening methods.

Once such agents or drugs are identified, they may be further tested inone or more biological assays to determine if they inhibit at least oneERM-associated function, such as proliferation.

Any ERM binding agents may be suitable for diagnostic use, especiallywhen the binding reaction to detect and/or quantitate ERM proteins iscarried out in vitro. Some or all of these binding agents may also besuitable for use as therapeutic agents to treat and/or prevent anERM-associated condition in vivo.

For example, any agents that are identified as ERM-binding agent may befurther assessed for their ability to inhibit cancer cell invasion in aMatrigel assay, which was used to demonstrate that anti-ezrin monoclonalantibody can inhibit cancer cell invasion. Alternatively, theERM-binding agent may be tested in an animal model for their ability toinhibit cancer metastasis in vivo. For instance, immuno-compromisedanimals (e.g., nude mice) may be implanted subcutaneously orintra-peritoneally with a metastatic cancer cell line (e.g., tumorxenograph implant into nude mice), and the ERM-binding agent is thenadministered to the animal to determine if it significantly inhibitscancer growth at the implant site (the primary site), and/orsignificantly reduces metastasis to a distant organ (such as lung).

Candidate binding agents to be assessed for their ability to bind atarget protein, and/or ability to inhibit a function of the targetprotein (e.g., inhibit ERM-associated proliferation, invasion ormetastasis) can be any chemical in nature (e.g., element, molecule,compound, drug), either made synthetically, or made by recombinanttechniques or isolated from a natural source, or a combination thereof.For example, test agents can be peptides, polypeptides, peptoids,sugars, hormones, or nucleic acid molecules. In addition, test agentscan be small molecules or molecules of greater complexity made bycombinatorial chemistry, for example, and compiled into libraries. Theselibraries can comprise, for example, alcohols, alkyl halides, amines,amides, esters, aldehydes, ethers and other classes of organiccompounds. Test agents can also be natural or genetically engineeredproducts isolated from lysates or growth media of cells—bacterial,animal or plant—or can be the cell lysates or growth media themselves.Presentation of test compounds to the test system can be in either anisolated form or as mixtures of compounds, especially in initialscreening steps.

In one embodiment of an assay to identify a test compound that binds toa target protein, the target protein may be immobilized on a solidsupport. The immobilized protein is then contacted with either a labeledtest compound (e.g., direct binding assay), or a labeled known targetprotein-binding agent plus a test compound or a group of test compounds(e.g., a competitive assay). The amount of labeled is then determined.The amount of label is proportional (in the direct assay) or inverselyproportional (in the competitive assay) to the ability of the testcompound to bind the target protein.

The label used can be, for example, a radioactive isotope, or afluorescent or colormetric label. The solid support can be any suitablesolid phase or matrix, such as resin in a packed column, suspended beadsin a solution, the wall of a plate or other suitable surface (e.g., awell of a microtiter plate), BIOCORE binding surface, column pore glass(CPG) or a pin that can be submerged into a solution, such as in a well.Linkage of the target protein to the solid support can be either director through one or more linker molecules.

In one embodiment, an isolated or purified target protein can beimmobilized on a suitable affinity matrix by standard techniques, suchas chemical cross-linking, or via an antibody raised against theisolated or purified protein, and bound to a solid support. The matrixcan be packed in a column or other suitable container and is contactedwith one or more compounds (e.g., a mixture) to be tested underconditions suitable for binding of the compound to the protein. Forexample, a solution containing compounds can be made to flow through thematrix. The matrix can be washed with a suitable wash buffer to removeunbound compounds and non-specifically bound compounds. Compounds whichremain bound can be released by a suitable elution buffer. For example,a change in the ionic strength or pH of the elution buffer can lead to arelease of compounds. Alternatively, the elution buffer can comprise arelease component or components designed to disrupt binding of compounds(e.g., one or more ligands or receptors, as appropriate, or analogsthereof which can disrupt binding or competitively inhibit binding oftest compound to the protein).

Fusion proteins comprising all of, or a portion of, a target proteinlinked to a second moiety not occurring in the target protein as foundin nature can also be prepared for use in another embodiment of themethod. Suitable fusion proteins for this purpose include those in whichthe second moiety comprises an affinity ligand (e.g., an enzyme,antigen, epitope). The fusion proteins can be produced by inserting thetarget ERM protein or a portion thereof into a suitable expressionvector which encodes an affinity ligand. The expression vector can beintroduced into a suitable host cell for expression. Host cells aredisrupted and the cell material, containing fusion protein, can be boundto a suitable affinity matrix by contacting the cell material with anaffinity matrix under conditions sufficient for binding of the affinityligand portion of the fusion protein to the affinity matrix.

Thus in one aspect of this embodiment, a fusion protein can beimmobilized on a suitable affinity matrix under conditions sufficient tobind the affinity ligand portion of the fusion protein to the matrix,and is contacted with one or more compounds (e.g., a mixture) to betested, under conditions suitable for binding of compounds to thereceptor or ligand protein portion of the bound fusion protein. Next,the affinity matrix with bound fusion protein can be washed with asuitable wash buffer to remove unbound compounds and non-specificallybound compounds without significantly disrupting binding of specificallybound compounds. Compounds which remain bound can be released bycontacting the affinity matrix having fusion protein bound thereto witha suitable elution buffer (a compound elution buffer). In this aspect,compound elution buffer can be formulated to permit retention of thefusion protein by the affinity matrix, but can be formulated tointerfere with binding of the compound(s) tested to the receptor orligand protein portion of the fusion protein. For example, a change inthe ionic strength or pH of the elution buffer can lead to release ofcompounds, or the elution buffer can comprise a release component orcomponents designed to disrupt binding of compounds to the receptor orligand protein portion of the fusion protein (e.g., one or more ligandsor receptors or analogs thereof which can disrupt binding of compoundsto the receptor or ligand protein portion of the fusion protein).Immobilization can be performed prior to, simultaneous with, or aftercontacting the fusion protein with compound, as appropriate. Variouspermutations of the method are possible, depending upon factors such asthe compounds tested, the affinity matrix selected, and elution bufferformulation. For example, after the wash step, fusion protein withcompound bound thereto can be eluted from the affinity matrix with asuitable elution buffer (a matrix elution buffer). Where the fusionprotein comprises a cleavable linker, such as a thrombin cleavage site,cleavage from the affinity ligand can release a portion of the fusionwith compound bound thereto. Bound compound can then be released fromthe fusion protein or its cleavage product by an appropriate method,such as extraction.

In some cases, one or more compounds can be tested simultaneously. Wherea mixture of compounds is tested, the compounds selected by theforegoing processes can be separated (as appropriate) and identified bysuitable methods (e.g., PCR, sequencing, chromatography). Largecombinatorial libraries of compounds (e.g., organic compounds, peptides,nucleic acids) produced by combinatorial chemical synthesis or othermethods can be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl.Acad. Sci. USA 90: 10922-10926, 1993; and DeWitt, S. H. et al., Proc.Natl. Acad. Sci. USA 90: 6909-6913, 1993, relating to tagged compounds.See also, Rutter, W. J. et al., U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible. Where compounds do not carrytags, chromatographic separation, followed by mass spectrophotometry toascertain structure, can be used to identify individual compoundsselected by the method, for example.

The instant invention also provides a method to identify agents thatinhibit one or more ERM family proteins. In this embodiment, a pluralityof agents are first identified as antagonists to at least one targetprotein using any of the above described methods. Then these antagonistsmay be further tested, using any of the above methods, for their abilityto inhibit a second (or third, etc.) family protein. Alternatively, themethod can be carried out in an array format, in that a number ofrelated target proteins may be tested simultaneously against one or moreindividual test compound(s) (such as immobilizing the target ERMs on anarray, and contacting the array with one or more test compounds). Ifeach test compounds is labeled by a unique identifiable tag, the typesand/or amounts of each tagged compound bound to each target proteins onthe array can be simultaneously determined.

Inhibitors in the present invention can also be designed by usingmolecular modeling. A computer model of a target protein or a closehomolog thereof may be used to identify any compounds that might bindthe target protein in the ligand binding sites. Alternatively,antagonistic compounds mimicking the natural ligands of these targetsmight be designed in silica. Alternatively, the nature of the inhibitorysequence can be determined by calculation, based on knowledge of areceptor or binding pocket. Other calculational strategies will be knownto those skilled in the art. Calculations such as these can be usefulfor directing the synthesis of inhibitors of the present invention in atime- and material-efficient manner, before actual synthesis andscreening techniques begin.

Other methods that can be adapted for screening the binding agents ofthe present invention are well known in the art, independent of the useof computer modeling. The use of peptide libraries is one way ofscreening large numbers of polypeptides at once. In one screening assay,the candidate peptides are displayed on the surface of a cell or viralparticle, and the ability of particular cells or viral particles to binda target ERM protein is detected in a “panning assay.” For instance, thegene library can be cloned into the gene for a surface membrane proteinof a bacterial cell, and the resulting chimeric polypeptide detected bypanning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).

In an alternate embodiment, the peptide library to be screened isexpressed as chimeric polypeptides on the surface of a viral particle.For instance, in the filamentous phage system, foreign peptide sequencescan be expressed on the surface of infectious phage, thereby conferringtwo significant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of infection. The group of almostidentical E. coli filamentous phages M13, fd, and fl are most often usedin phage display libraries, as either of the phage gIII or gVIII coatproteins can be used to generate chimeric polypeptides withoutdisrupting the ultimate packaging of the viral particle (Ladner et al.,WO 90/02809; Garrard et al., WO 92/09690; Marks et al., J. Biol. Chem.267: 16007-16010, 1992; Griffiths et al., EMBO J 12: 725-734, 1993;Clackson et al., Nature 352: 624-628, 1991, and Barbas et al., PNAS 89:4457-4461, 1992).

The field of combinatorial peptide libraries has been reviewed (Gallopet al. J. Med. Chem. 37: 1233-1251, 1994), and additional techniques areknown in the art (Gustin, K Virology 193: 653-660, 1993; Goeddel et al.U.S. Pat. No. 5,223,408; Markland et al. PCT publication WO92/15679;Bass et al. Proteins: Structure, Function and Genetics 8: 309-314, 1990;Cunningham, B. C. Science 247: 1461-1465, 1990; Lowman, H. B.Biochemistry 30: 10832-10838, 1991; Fowlkes et al. U.S. Pat. No.5,789,184; Houghton, Proc. Natl. Acad. Sci. U.S.A. 82: 5131-5135, 1985)for generating and screening peptide libraries.

U.S. Pat. No. 6,420,110 (incorporated herein by reference) discloses amethod for isolating biologically active peptides. Using the techniquesdisclosed therein, a polypeptide ERM protein inhibitor of the presentinvention may be developed, which interacts with a chosen ERM protein,and inhibits the function thereof. The inhibition can be readily testedin many suitable in vitro or in vivo models, such as those describedherein.

In a representative example, this method is utilized to identifypolypeptide ERM protein antagonists which have antagonistic activitywith respect to one or more types of cells expressing at least one ERMprotein. One of skill in the art will readily be able to modify theprocedures outlined below to find polypeptides with any desiredactivity. In the example, in the display mode, the chimeric polypeptidelibrary can be panned with the target cells or immobilized targetprotein in order to enrich for polypeptides which bind to that cell orreceptor. At that stage, the polypeptide library can also be pannedagainst one or more control cell lines (that does not express any of thetarget proteins) in order to remove polypeptides which bind the controlcells. In this manner, the polypeptide library which is then tested inthe secretion mode can be enriched for polypeptides which selectivelybind target cells (relative to the control cells). Thus, for example,the display mode can produce a polypeptide library enriched forpolypeptides which preferentially bind ERM-expressing tumor cellsrelative to normal cells, or any other differential bindingcharacteristic.

In the secretion mode, the polypeptides are tested for antiproliferativeand/or invasive activity against the target cell, using any of a numberof techniques known in the art. For instance, BrdU or other nucleotideuptake can be measured as an indicator of proliferation. Matrigelinvasion assay may be used to test invasiveness. Animal models (e.g.,tumor xenograph in nude mice) may be used to test metastasis, etc. Othersuitable functional test for specific ERM proteins are well-known in theart. Furthermore, the secretion mode can include negative controls inorder to select for polypeptides with specific biological activity(e.g., antiproliferative/anti-invasiveness/anti-metastatic activity),rather than non-specific effects such as general toxicity.

Exemplary ERM-Interacting Proteins

As described above, any ERM binding proteins must contain a domain,motif or moiety (e.g., a peptide fragment) that binds ERM. SuchERM-binding peptides may interfere with ERM protein function, and thusserve as candidate molecules for testing their ability as ERMantagonists. Some of the known ERM binding proteins are listed below.Other ERM binding proteins may be readily identified using anyart-recognized techniques for identifying protein-protein interaction,such as various kinds of interaction-trap assays (e.g., yeast two-hybridassays), phage display, etc.

Bonilha and Rodriguez-Boulan (Invest. Ophthal. Vis. Sci. 42: 3274-3282,2001) identified EBP50 and SAP97 as binding partners for ezrin, anactin-binding protein crucial for morphogenesis of apical microvilli andbasolateral in foldings in retinal pigment epithelial (RPE) cells.Immunofluorescence microscopy detected a polarized distribution of EBP50at apical microvilli and of SAP97 at the basolateral surface of RPEcells, which overlapped with ezrin.

By two-hybrid analysis, affinity precipitation, and mutation analysis,Mykkanen et al. (Molec. Biol. Cell 12: 3060-3073, 2001) determined thatthe alpha-helical region of ezrin interacted with the C-terminal Igdomains of the microfilament-associated protein palladin. Thepalladin-binding site was masked in dormant wild-type ezrin. By doublestaining of ezrin and palladin in several cell lines, Mykkanen et aL(supra) found that the subcellular localization of ezrin differedbetween epithelia and smooth muscle cells. In epithelial cells, such asHeLa, ezrin localized at the cortical actin skeleton and demonstratedlittle overlap with palladin. However, in intestinal smooth musclecells, ezrin demonstrated a filamentous staining pattern and partialco-localization with palladin.

Cheng et al. (J Neurosci. 25(2): 395-403, 2005) identified two regionson the neural cell adhesion molecule L1 as ERM-binding sites—the RSLEregion and a novel juxtamembrane ERM-binding region.

Morales et al. (Proc Natl Acad Sci USA. 101(51): 17705-10, 2004)indicated that the ERM-binding phosphoprotein 50/Na⁺/H⁺ exchangerregulatory factor 1 (EBP50/NHERF1) binds the N-terminal domain of ERMproteins. Terawaki et al. (Acta Crystallogr D Biol Crystallogr. 59(Pt1): 177-9, 2003) previously reported the crystal structure of thecomplexes between the radixin FERM domain and the C-terminal regions ofNHERF and NHERF2.

In vitro binding studies showed that radixin associates directly withthe carboxy-terminal cytoplasmic domain of human MRP2 (multidrugresistance protein-2). NF2 tumor suppressor protein and radixin alsointeracts with the carboxy-terminal domain of layilin, a cell surfacehyaluronan receptor (Bono et al., Exp. Cell Res. May 20, 2005,epublication).

Ivetic et al. (J. Biol Chem. 279(32): 33263-72, 2004) have not onlyidentified ezrin and moesin as binding partners of the 17-amino acidL-selectin tail, but also two basic amino acid residues within thatL-selectin tail as being required for binding to ezrin-radixinmoesin(ERM) proteins: arginine 357 and lysine 362. Serrador et al. (Eur JImmunol. 32(6): 1560-6, 2002) also found that a juxta-membrane aminoacid sequence of P-selectin glycoprotein ligand-1 (PSGL-1) is involvedin moesin binding.

Iwase et al. (J Biol Chem. 279(12): 11898-905, 2004) found that thecytoplasmic domain of Neutral endopeptidase 24.11 (NEP) contains apositively charged amino acid cluster, which binds the N terminalfragment of ezrin/radixin/moesin (ERM) proteins. Binding of ERM proteinsto NEP results in decreased binding of ERM proteins to the hyaluronanreceptor CD44, a main binding partner of ERM proteins. Moreover, cellsexpressing wild-type NEP demonstrate decreased adhesion to hyaluronicacid and cell migration.

Hamada et al. (EMBO J. 22(3): 502-14, 2003) reported the crystalstructure of the radixin FERM (4.1 and ERM) domain complexed with theICAM-2 cytoplasmic peptide. The non-polar region of the ICAM-2 peptidecontains the RxxTYxVxxA sequence motif to form a beta-strand followed bya short 3(10)-helix. It binds the groove of the phosphotyrosine-binding(PTB)-like subdomain C mediated by a beta-beta association and severalside-chain interactions. The binding mode of the ICAM-2 peptide to theFERM domain is distinct from that of the NPxY motif-containing peptidebinding to the canonical PTB domain. Mutation analyses based on thecrystal structure reveal the determinant elements of recognition andprovide the first insights into the physical link between adhesionmolecules and ERM proteins.

Serrador et al. (J Biol Chem. 277(12):10400-9, 2002) also reported aphosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3and the amino-terminal domain of ERM proteins, and the role of specificserine residues (Ser487 and Ser489, possibly also Ser496) within thecytoplasmic region of ICAM-3 for its ERM-directed positioning at thetrailing edge of motile lymphocytes.

Hamada et al. (Acta Crystallogr D Biol Crystallogr. 57(Pt 6): 889-90,2001) reported the crystal structure of RhoGDI complexed with the FERMdomain of radixin. Takahashi et al. (Oncogene 16(25): 3279-84, 1998)showed that the N-terminal region of radixin furthermore interacts withDbl, a stimulatory GDP/GTP exchange protein of the Rho family members.This interaction does not affect the Dbl activity to stimulate theGDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbldoes not interact with radixin which is precomplexed with Rho GDI, andRho GDI displaces Dbl from radixin.

Yonemura et al. (J Cell Biol. 140(4): 885-95, 1998) reported that ERMproteins bind to a positively charged amino acid cluster in thejuxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2.

Methods of Treatment

In certain embodiments, the present invention provides methods oftreating an individual suffering from a proliferative condition, such ascancer, through administering to the individual a therapeuticallyeffective amount of an ERM binding agent as described above.

In other embodiments, the invention provides methods of preventing ordelaying the onset, and/or retarding the progression of theproliferative condition (e.g., cancer) in an individual throughadministering to the individual a therapeutically effective amount of anERM binding agent. These methods are particularly aimed at therapeuticand prophylactic treatments of animals, and more particularly, humans.In these embodiments, the ERM binding agent binds an ERM protein andinhibits at least one function of the ERM protein.

In certain embodiments of such methods, one or more ERM binding agentscan be administered, together (simultaneously) or at different times(sequentially). In addition, ERM binding agents can be administered withone or more other compounds for treating the proliferative condition(e.g. cancer). The two or more compounds may be administeredsimultaneously or sequentially.

Methods of the present invention can be used to treat a variety ofproliferative conditions, including cancer and (benign) proliferativedisorders.

The cancers that can be treated using the subject method include, butare not limited to: ovarian cancer, endometrial cancer, breast cancer,glioblastoma, schwannoma, meningioma, malignant mesothelioma,neurofibromatosis, colon cancer, oral cancer, or a cancer selected fromthe group consisting of: lung cancer, prostate cancer, pancreaticcancer, leukemia, liver cancer, stomach cancer, uterine cancer,testicular cancer, brain cancer, non-hodgkin's lymphoma, hodgkin'slymphoma, Ewing's sarcoma, osteosarcoma, neuroblastoma,rhabdomyosarcoma, melanoma, and brain cancer.

The cancer may be invasive and/or metastatic.

The benign proliferative disorders that can be treated using the subjectmethod include, but are not limited to: tuberosclerosis, psoriasis,endometriosis, or polyps (such as colon polyps).

Pharmaceutical Compositions

In certain embodiments, the ERM binding agents of the present inventionare formulated as a pharmaceutical composition with a pharmaceuticallyacceptable carrier or salt. The term “pharmaceutically acceptable salts”refers to physiologically and pharmaceutically acceptable salts of thecompounds of the invention, e.g., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto.

The ERM binding agents, when used as therapeutic agents, can beadministered alone or as a component of a pharmaceutical formulation(composition). The ERM binding agents may be formulated foradministration in any convenient way for use in human or veterinarymedicine. In certain embodiments, the ERM binding agents included in thepharmaceutical preparation may themselves be active, or may be prodrugs.The term “prodrug” refers to compounds which, under physiologicalconditions, are converted into therapeutically active agents (such as abinding agent that is normally inhibited before administration, but theinhibition is removed by enzymatic cleavage or pH change, etc., when thebinding agent is delivered in vivo to the individual).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Pharmaceutical compositions of the ERM binding agents include thosesuitable for oral/nasal, topical, parenteral and/or intravaginaladministration. The pharmaceutical compositions may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

Methods of preparing these pharmaceutical compositions or compositionsinclude combining an ERM binding agent and a carrier, and optionally,one or more accessory ingredients. In general, the pharmaceuticalcompositions can be prepared with a liquid carrier, or a finely dividedsolid carrier, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions for oral administration may be in the formof capsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of an artemisinin-related compound as an activeingredient. An artemisinin-related compound may also be administered asa bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more ERM bindingagents of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In particular, therapeutic agents or pharmaceutical compositions can betopically, either to skin or to mucosal membranes, such as those of thecervix and vagina. The topical pharmaceutical compositions may furtherinclude one or more of the wide variety of agents known to be effectiveas skin or stratum corneum penetration enhancers. Examples of these are2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to anartemisinin-related compound, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an ERM binding agents,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more ERM binding agents in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofone or more anti-tumor therapeutic agents in biodegradable polymers suchas polylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectablepharmaceutical compositions are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue.

Pharmaceutical compositions for intravaginal administration may bepresented as a suppository, which may be prepared by mixing one or morecompounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Optionally, such pharmaceutical compositions suitablefor vaginal administration also include pessaries, tampons, creams,gels, pastes, foams or spray pharmaceutical compositions containing suchcarriers as are known in the art to be appropriate.

Methods of Administration

In certain embodiments, the subject treatment methods of the inventioncan be used alone. Alternatively, the subject treatment methods may beused in combination with other conventional anti-proliferativetherapeutic approaches directed to treatment or prevention ofproliferative disorders (e g., tumors). For example, such methods can beused in prophylactic cancer prevention, prevention of cancer recurrenceand metastases after surgery, and as an adjunct to other conventionalcancer therapies. The present invention recognizes that theeffectiveness of conventional cancer therapies (e.g., chemotherapy,radiation therapy, phototherapy, immunotherapy, and surgery) can beenhanced through the use of an ERM binding agent which inhibits ERMprotein function.

A wide variety of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a ERM binding agent of the present invention is administered incombination with another conventional anti-neoplastic agent, eitherconcomitantly or sequentially, such therapeutic agent may enhance thetherapeutic effect of the anti-neoplastic agent or overcome cellularresistance to such anti-neoplastic agent. This may allow decrease ofdosage of an anti-neoplastic agent, thereby reducing the undesirableside effects, or restores the effectiveness of an anti-neoplastic agentin resistant cells.

Pharmaceutical compounds that may be used for such combinationchemotherapy include, merely to illustrate: aminoglutethimide,amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, vinorelbine, and HERCEPIIN® and other antibodytherapeutics.

These chemotherapeutic compounds may be categorized by their mechanismof action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunombicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (vascular endothelial growth factor (VEGF) inhibitors,fibroblast growth factor (FGF) inhibitors); angiotensin receptorblocker, nitric oxide donors; anti-sense oligonucleotides; antibodies(trastuzumab); cell cycle inhibitors and differentiation inducers(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,hydrocortisone, methylpednisolone, prednisone, and prenisolone), growthfactor signal transduction kinase inhibitors; mitochondrial dysfunctioninducers and caspase activators; and chromatin disruptors.

Depending on the nature of the proliferative disorder and the therapy,administration of the ERM binding agents of the invention may becontinued while the other therapy is being administered and/orthereafter. Administration of the ERM binding agents may be made in asingle dose, or in multiple doses. In some instances, administration ofthe ERM binding agents is commenced at least several days prior to theconventional therapy, while in other instances, administration is beguneither immediately before or at the time of the administration of theconventional therapy.

In certain therapeutic applications, the ex vivo-derived inhibitors areutilized in a manner appropriate for therapy in general. For suchtherapy, the inhibitors or vectors encoding inhibitors of the inventioncan be formulated for a variety of modes of administration, includingsystemic and topical or localized administration. In such embodiments, apolypeptide inhibitor may be combined with a pharmaceutically acceptableexcipient, e.g., a non-pyrogenic excipient. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection beingpreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous injection, the inhibitors of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theinhibitors may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the peptides are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, particularly cosmetic pharmaceutical compositions, theoligomers of the invention are formulated into ointments, salves, gels,or creams as generally known in the art.

Alternative means of administration of peptides have been developed.Sustained-release pharmaceutical compositions (Putney, et al. NatureBiotechnology 1998, 16, 153-157) are advantageous, requiring feweradministrations and, often, lower dosages. Techniques for oral deliveryof peptides have been reviewed (Fasano, A. Trends in Biotechnology 1998,16, 152-157), as have several site-specific means of peptide delivery(Pettit, D. K. et al. Trends in Biotechnology 1998, 16, 343-349).Additional techniques for therapeutic administration of peptides areknown to those of skill in the art.

The teachings of all publications and patents cited herein areincorporated herein by reference.

EXAMPLES

The following examples are for illustrative purpose only, and should inno way be construed to be limiting in any respect of the claimedinvention.

Example 1 Immuno-Responsive ERM Protein is Detected in Ascitic Fluidfrom Ovarian Cancer Patients

To determine if ERM proteins are shed from the surface of diseased cellsand appear in body fluids, ascitic fluid from four patients withmetastatic OVCA were obtained by paracentesis. The samples wereimmediately centrifuged, and the supernatants were snap frozen in liquidnitrogen. The samples were stored in the Yale Discovery to Cure tissueand fluid bank for an extended period of time, before they were thawedand diluted 6-8 fold (because of overloading by neat samples), andstudied by Western blotting using anti-ezrin antibody (FIG. 8).

It was readily seen from the Western blot that all patients' asciticfluids contained large amounts of immuno-reactive ezrin (ir-ezrin).

Example 2 Ezrin Expression is Correlated with Cancer Progression

Using Immunohistochemistry, it was found that ezrin is over-expressed inOVCA cells but not in normal ovary or in the superficial ovarianepithelial cells. In addition, this expression level was much higher inascitic cells from patients with metastatic OVCA (data not shown). Thestaining of ir-ezrin was found at the base of protuberances and alongthe cytoplasmic edge of the ruffles, and also at the intercellularbridges. All of these stainings are characteristic of the role of ezrinin cell membrane specialization.

This experiment indicates that ezrin expression is correlated withcancer progression, and ERM protein expression is higher in cancer cellsthan in normal cells, and highest in metastatic cancer cells.

Western blot analysis also confirmed this finding. The relativeexpression level of ezrin was measured in protein samples from OVCApatients at different stages of cancer progression (e.g., primary cancervs. metastatic cancer), as compared to normal ovary (FIG. 3).

For a typical Western blot analysis, approximately 200 mg of tissueskept frozen at −80° C. are homogenized and then lysed at 4° C. using 0.5ml of ice-cold RIPA buffer (0.1% SDS, 1% Triton X-100, 1% deoxycholate,0.15 M NaCl, 2 mM EDTA, 25 mM Tris, pH 7.5) containing 4 mg/ml ofprotease inhibitor cocktail tablet (Boehringer-Mannheim, Indianapolis,Ind., USA). The lysates are centrifuged at 105,000×g for 30 min at 4° C.into particulate and cytosolic fractions. The particulate pellet isresuspended in the same volume as cytosolic fractions. The equal amountof protein (26 μg) from each sample is subjected to 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferredonto polyvinyl diflouride membranes (Millipore, Bedford, Mass., USA) ina solution containing 25 mM Tris, 92 mM glycine, 0.01% SDS, and 20%methanol. After blocking with 6% skim milk, the membranes are probedwith B22 or β-tubulin (Vector Labs) for 1 h at room temperature.β-Tubulin (or other proteins such as actin) may be used as an internalcontrol. The immunoreactive proteins are visualized usingperoxidase-conjugated secondary antibodies (Vector Labs) and theECL-chemiluminescence Western blot analysis kit (Amersham, ArlingtonHeights, Ill., USA). Protein quantitation is determined by theBicinchoninic acid protein assay kit (Pierce, Rockford, Ill., USA).Analyses are performed in five independent series. The signal intensityof each band is analyzed using NIH Image software. The expression levelsare determined as ratios of band intensity between ezrin and thereference protein (β-tubulin in this case) to correct for variation inthe amounts of protein, and are statistically compared by Mann-WhitneyU-test.

It was clear that ezrin expression was much higher (e.g., at leastthree-fold higher) in primary OVCA than in normal ovary, and even higher(e.g., close to four-fold higher) in metastatic OVCA. There was a clearcorrelation between ezrin expression and cancer progression. Thedifferences were statistically significant.

Example 3 ERM Antibody Inhibits Matrigel Invasion by MetastaticEndometrial Carcinoma Cells in a Dose-Dependent Manner

It has been previously shown that ezrin antisense polynucleotidesinhibited invasion of highly-metastatic endometrial carcinoma cells inthe Matrigel membrane cell invasion assay in proportion to ezrinexpression, although those antisense polynucleotides did not appear toaffect cancer cell proliferation. It is shown herein that ezrin antibodyhad surprisingly the same effect as the ezrin antisense polynucleotidesin inhibiting cell invasion in the Matrigel assay (FIG. 4A).

In one experiment, an antigen-affinity-purified rabbit antiserum(polyclonal antibody “B22”) to human placental ezrin was used, which wasobtained as a gift from Dr Anthony Bretscher (Cornell University,Ithaca, N.Y., USA) (Khanna et al., Cancer Res. 61: 3750-3759, 2001). B22clearly inhibited ENDOCA cell invasion in the Matrigel assay (FIG. 4A).This antiserum does not recognize the related proteins moesin andradixin (Franck et al., J. Cell Sci. 105: 219-231, 1993).

The same results for Matrigel penetration were also obtained using acommercially available antibody from Sigma (Product No. E 8897,Sigma-Aldrich, St. Louis, Mo.), in both Ishikawa cells (ENDOCA cells)and SKOV3 cells (an OVCA cell line, see Examples 4 and 7 below). Theinhibitory effect of ezrin antibodies on cell growth was also seen inthese experiments (see FIG. 6). In addition, the use of different titerswas performed. All results were dose-responsive (see FIG. 6).

Specifically, cancer cells incubated with the B22 antibody showeddiminished ability to penetrate the Matrigel, such that by Day 2 of theassay, the difference between the B22 treated sample and the controlsamples are statistically significant (FIG. 4A).

Incubating the highly metastatic cancer cells at the presence of as lowas 2.5 μg/mL of the B22 antibody was sufficient to cause thestatistically significant difference in this assay (FIG. 4B).

Importantly, it is clearly shown herein that the anti-invasive effect ofanti-ezrin antiserum is dose-dependent (FIG. 4B). In FIG. 5, ananti-ezrin monoclonal antibody was diluted 1:100, 1:500, or 1:1000,before it was used in the Matrigel invasion assay using OVCA cells. Aclear dose-dependent response was observed compared to the controlassay.

These experiments unequivocally demonstrated the surprising finding thatERM binding agents (e.g., antibodies), when administered to livingcells, can inhibit the function of their target molecules in adose-dependent manner, despite the fact that such target molecules haveno extracellular domain.

For a typical Matrigel invasion assay, cells are cultured for 24 hrswith serum-free OptiMEM 1×. A 0.5 ml suspension of 2.5×10⁴ cells is thenlayered in the upper compartment of a Boyden Chamber (BD Bioscience,Bedford, Mass., USA). The chamber has a polycarbonate filter (8 μm poresize) pre-coated with 20 μg of Matrigel basement membrane. The cells arethen incubated for a further 24 h at 37° C. with serum-free OptiMEM 1×.Other reagents, such as antibodies or other inhibitors, stimulators maybe incubated with the cells to test their ability to affect Matrigelinvasion.

Cells that are motile and invade through the Matrigel membrane into thelower chamber are counted, and their numbers compared with atimed-vehicle control group. At the end of the incubation the Matrigelis scraped away and the remaining plastic filters are harvested andstained with the Diff-Quick stain set (DADE HEHRING AG, Dudingen,Switzerland) in order to count the cells that have penetrated theMatrigel membrane. All incubations are done in triplicate. Allexperiments are performed at least three times.

The Ishikawa cell line used in this study is an estrogen-dependent, welldifferentiated endometrial adenocarcinoma cell line. It was a gift fromDr. Nishida, Department of Obstetrics and Gynecology, Tsukuba UniversitySchool of Medicine (Nishida et al., Acta Obstet. Gynaecol. Jpn. 37:1103-1111, 1985). An estrogen independent metastatic subclone of theIshikawa line (mEIIL: metastatic Estrogen-Independent Ishikawa Line) wasestablished in the Department of Obstetrics and Gynecology, NihonUniversity School of Medicine, by culturing Ishikawa cells inestrogen-free medium over 400 days, followed by in vivo clonal selection(Sakamoto et al., Acta Obstet. Gynaecol. Jpn. 47: 249-256, 1995).Ishikawa and mEIIL cells were maintained in Eagle's MEM (basal medium;Sigma Chemical Co., St. Louis, Mo.) supplemented with 10%heat-inactivated fetal calf serum (FCS, Gibco BRL, Grand Island, N.Y.)and 1% penicillin/streptomycin/fungizone (Sigma Chemical Co.) at 37° C.in a humidified 5% CO₂ atmosphere. The culture medium was changed every7 days. Four days prior to the experiments, cell cultures were changedfrom basal medium to phenol red-free mixture of Ham's F-12 andDulbecco's modified Eagle's medium (Sigma Chemical Co.) supplementedwith 5% FCS stripped of steroids by a dextran-coated charcoal treatment(experimental medium). Two days later, the cells were plated into 9 cmdishes (Corning, Corning, N.Y.) and used for all experiments.

Both mEIIL cells and Ishikawa cells were similarly tested in this assay,and both yielded similar results. The three negative controls for eachexperiment included no antibody control, anti-rabbit antisera control,and anti-human antisera control. Statistically significant differencesare represented by “*” (P <0.05) or “**” (P<0.01).

Example 4 ERM Antibody Inhibits OVCA Cell Proliferation

To demonstrate the effect of ERM antibodies on OVCA proliferation,unaggregated cells were suspended by passing the cells through a 21Gneedle. To obtain cell adherence on the pre-coated plastic dishes, 200μl of cell suspension (5×10³-5×10⁴ cells) was pipetted into each well ofa 96 well flat-bottomed micro-plate (Becton-Dickinson scientific, NJ)and incubated for 48 h. SKOV3 cells were cultured in McCoy medium(supplemented with 10% FBS) while DOV13 and OVCA ascites cells werecultured in MEM (supplemented with 10% FBS). There was then an optional24 h pre-incubation with serum-free OptiMEM 1×. The medium was changedto that containing various dilutions of ERM antibodies or other testagents for an additional 24-48 h.

The living cell mass was determined using the MTT assay as previouslydescribed (Song et al., J. Clin. Invest. 106: 1209-1220, 2000; Song etal., Mol. Hum. Reprod. 8: 447-455, 2002): 20 μl of MTT{3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, StLouis) dye was added to each well 4 h before the end of the incubation.The wells were decanted, and 100 μl of acidified isopropyl alcohol wasadded to solubilize the reactive crystals. An automatic micro-platereader (Model 550; Bio-Rad, Hercules, Calif.) was used to measureabsorbency at 540 nm. All the tests were accompanied by a Cell Titerassay (CellTiter 96 Aqueous, Promega, Madison, Wis., USA) following themanufacture's manual.

FIG. 6 shows that anti-ezrin mAb also inhibits OVCA cell growth. (e.g.,increase in cell number) by the same starting cells that are in theabove figures. These cells are grown separately, in flasks, to assesscell growth or increase in cell number. The number of cells in theflasks at the end of a 24-hour period are counted. The results fromcells treated with 1:1000 or 1:500 dilutions of anti-ezrin mAb arecompared with that of the control untreated cultures. At the end of theexperiment, the control group had four times as many cells as the 1:500dilution of anti-ezrin mAb (e.g., about 75% inhibition). The decrease incell number in mAb-treated culture may be due to inhibited cellproliferation or increased apoptosis, or both.

Example 5 ERM Protein Antibody Binds to OVCA Cells in Culture

Results also demonstrated that exposure of ezrin-expressing ovariancancer cells to a commercially available mAB against recombinant ezrinresults in the binding of the mAB to live cells (FIG. 7). Controlanti-actin (intracellular protein) mAB (negative control) showed onlyoccasional binding to the cells, and control anti-FAS Receptor (cellsurface antigen) mAB (positive control) bound well to the cells,indicating that the study is a proper demonstration of the binding ofanti-ezrin mAB to the ezrin in the area of the cell membrane. Thesestudies are therefore confirmatory of the finding that antiserum B22blocked ezrin action in cancer cells.

In this experiment, OVCA cells in culture were exposed anti-exrin mAbfor only 5 minutes to avoid endocytosis of the mAb. The cells were thenwashed, fixed, and stained using the appropriate detection antibodiesand the DAB reaction.

Example 6 ERM Proteins are Present in Cell Membranes and in Fragments ofEzrin-Positive Cells

Electron microscopy was used to determine definitively whether theanti-ezrin mAB was bound to a membrane or an extracellular site. Whilestudies showed the presence of free-floating, ezrin-positive material,the intact cells only have intracellular ezrin. Furthermore, followingadministration to the cultures, the living cells took up the anti-ezrinantibody, which was only found in the actual membranes (see above andFIG. 7). These results further indicate that ezrin is restrained tobeing in or near the surface of the membrane, and this is where theantibodies encounter ezrin. On the other hand, the presence of their-ezrin-positive fragments explains the presence of ezrin in bodycavity fluids, and indicates that ezrin may be founds in body fluids inaddition to ascitic fluid. These findings indicate that ERM proteins mayserve as clinical tumor markers.

Example 7 ERM Antibodies Inhibit Tumor Xenograph Invasion/Metastasis

Immuno-compromised SCID mice are injected with about 5×10⁶ SKOV3 OVCAcells intraperitoneally to allow cancer cells to proliferate andmetastasize. It has been previously shown that commercially availableanti-ezrin antibodies can inhibit SKOV3 cell proliferation, as well asMatrigel invasion in the Matrigel penetration assay.

The SKOV3 cells are purchased from American Type Culture Collection(ATCC, Rockville, Md., USA). Prior to study, cells are maintained inMcCoy medium or minimal essential medium (MEM) supplemented withpenicillin, streptomycin, and 10% fetal calf serum (GIBCO BRL,Gaithersburg, Md., USA). Cells are cultured in 5% CO2 and humidified airand the medium was changed twice weekly. After flask-incubation tosub-confluence (60-70%) in McCoy medium or MEM containing 10% FBS, themedium is changed to phenol red-free, serum-free OptiMEM 1×.

One group of injected mice are used as control, and receive only controlvehicle injection. The other similarly injected mice (the experimentalgroup) are treated with injections of species compatible anti-ezrin mAb.Animals are assessed for rate of implantation and amount of tumor growthusing standard methods starting four days after the injection of cancercells to determine whether the treated mice have less and slower cancergrowth, with fewer or no distant metastasis in the lung compared to thecontrol mice.

A full mAb dose-response curve is also obtained to facilitate thedetermination of the right dose of antibody to be used in the followingexperiments.

The selected dose of mAb (from above) is then used to treat establishedOVCA. This is done by breeding transgenic mice that express the SV40large T antigen under the transcriptional control of a portion of the 5′upstream region (5′-UTR) of the Mullerian Inhibitory Substance type IIreceptor (MSIIR) gene (the TgMISIIR-TAg mice). It is expected that about50% of the females spontaneously develop an ovarian epithelial carcinomathat is similar in histology and in its metastatic route to human OVCA.These tumors also over-express ezrin, which is confirmed by WesternBlotting.

The mice are observed for the presence of tumor mass using MRI, and thentreated by intra-peritoneal anti-ezrin mAb treatment. Control group micereceive vehicle treatment only. The animals normally expire within fourweeks of the presence of demonstrable tumor, and thus they aresacrificed at either four weeks or six weeks. Tumor burden and micelongevity are assessed at the end point.

The female TGMISIIR-TAg mice develop bilateral ovarian tumors withspread to peritoneal organs and the presence of ascites. They typicallysuccumb to disease with an average latency of 140 days. Thus thesefemales are infertile. Thus to breed this line of transgenic mice, it isnecessary to mate the male transgenic TgMISIIR-TAg mice with wild typefemales to create a breader clone.

Example 8 Sandwich Assay for ERM protein Detection/Quantitation

A sandwich ELISA assay is used to detect and/or quantitate ERM proteinin tissue sample/fluids. For example, to detect/quantitate ezrin in asample, binding agents such as an ezrin capture antibody is bound to a96-well plastic plate (or other solid support). Ezrin in samples is thencaptured and then detected/quantitated by a specific antibody. The thirdelement in the “sandwich” is a species-specific anti-IgG that is labeledwith an enzyme, such as peroxidase. The peroxidase reaction is developedand quantitated by an ELISA plate reader.

Samples for this assay may be furnished from any source, such as by theYale University “Discovery to Cure” cancer specimen bank. Recovery ofadded ascitic fluid ir-ezrin may also be preformed.

Following standardization and establishment of reliability criteria, theassay is applied to ascitic fluid obtained at diagnostic abdominalendoscopy for infertility of unknown origin (“negative control”).Further testing may be performed on fluids and blood from patientsundergoing surgery or radiation/chemotherapy for OVCA/PPC and using thesame materials from the Yale “Discovery to Cure” cancer specimen bank. Asimilar strategy will be employed for other cancers and ezrin-relateddiseases.

The Ovarian Cancer Tissue Bank (OCTB) is located in the Department ofObstetrics, Gynecology, and Reproductive Sciences of Yale University,and contains approximately 500 tissue samples of primary and metastaticovarian cancers as well as tissue samples from normal ovaries. Inaddition, as part of the NCI Ovarian Cancer Detection Program, thefacility has in storage ascites and serum samples from patients withovarian cancer and normal age matched controls. An important componentof the Tissue Bank is its panel of ovarian cancer cells (n=36) isolatedfrom ascites and 10 immortalized normal Ovarian Surface Epithelial cells(OSE). The panel is regularly used for the screening of new compoundsthat may have cytotoxic effects on ovarian cancer.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific method and reagents described herein, including alternatives,variants, additions, deletions, modifications and substitutions. Suchequivalents are considered to be within the scope of this invention andare covered by the following claims.

1. A method of inhibiting a proliferative condition in an individual,comprising administering to the individual an effective amount of abinding agent which binds an ERM family protein.
 2. The method of claim1, wherein the proliferative condition is cancer.
 3. The method of claim2, wherein the cancer is ovarian cancer, endometrial cancer (endometrialadenocarcinoma, such as UEC), primary peritoneal cancer (PPC), renaladenocarcinoma, brain hemangioblastoma, pancreatic adenocarcinoma,epidermoid carcinoma, osteosarcoma, epithelial cancer, melanoma,squamous skin carcinoma, leukemia, breast cancer, glioblastoma,schwannoma, meningioma, malignant mesothelioma, neurofibromatosis, coloncancer, oral cancer, or rhabdomyosarcoma. 4-6. (canceled)
 7. The methodof claim 1, wherein the proliferative condition is a benignproliferative disorder.
 8. The method of claim 7, wherein theproliferative condition is tuberosclerosis, psoriasis, endometriosis,complex endometrial hyperplasia (cH), atypical endometrial hyperplasia(aH), polyps, or neurofibromatosis.
 9. The method of claim 1, whereinthe individual is a human or a non-human mammal.
 10. The method of claim1, wherein the binding agent is an antibody, or a functional fragmentthereof. 11-16. (canceled)
 17. The method of claim 1, wherein thebinding agent inhibits the interaction of the ERM family protein with acell surface receptor.
 18. (canceled)
 19. The method of claim 1, whereinthe ERM family protein is ezrin.
 20. The method of claim 1, furthercomprising administering a second therapeutic agent.
 21. (canceled) 22.A method of diagnosis of a proliferative condition in an individual,comprising determining the amount and/or concentration of an ERM familyprotein in a body fluid sample from an individual suspected of having orat risk of having the proliferative condition, wherein an amount and/orconcentration of the ERM family protein significantly higher in the bodyfluid sample from the individual than in a normal or control sample isindicative of the existence of the proliferative condition in theindividual.
 23. A method of monitoring in an individual the progress orrecurrence of a proliferative condition, comprising determining theamount and/or concentration of an ERM family protein in a body fluidsample from an individual who has or had the proliferative condition,wherein an amount and/or concentration of the ERM family proteinsignificantly higher in the body fluid sample from the individual thanthat in a normal or control sample is indicative of the progress orrecurrence of the proliferative condition in the individual. 24-25.(canceled)
 26. The method of claim 22, wherein the amount and/orconcentration of the ERM family protein in the sample is proportionallyindicative of the severity and/or extent of the proliferative condition.27. The method of claim 22, wherein the amount and/or concentration ofthe ERM family protein is used along with the results of one or morediagnostic tests selected from the group consisting of: mammography, anearly mammography program, a frequent mammography program, a biopsyprocedure using a tissue of the individual, an ultrasound analysis of asuspected disease organ and optionally a normal organ, a magneticresonance imaging (MRI) analysis of a suspected disease organ andoptionally a normal organ, an electrical impedance (T-scan) analysis ofa suspected disease organ and optionally a normal organ, ductal lavage,a nuclear medicine analysis, sequence analysis of one or moredisease-associated genes, and a thermal imaging of a suspected diseaseorgan and optionally a normal organ.
 28. The method of claim 22, whereinthe proliferative condition is cancer.
 29. The method of claim 28,wherein the cancer is ovarian cancer, endometrial cancer (ENDOCA),endometrial adenocarcinoma (e.g., UEC), primary peritoneal cancer (PPC),renal adenocarcinoma, brain hemangioblastoma, pancreatic adenocarcinoma,epidermoid carcinoma, osteosarcoma, epithelial cancer, melanoma,squamous skin carcinoma, leukemia, breast cancer, glioblastoma,schwannoma, meningioma, malignant mesothelioma, neurofibromatosis, coloncancer, oral cancer, or rhabdomyosarcoma.
 30. (canceled)
 31. The methodof claim 22, wherein the proliferative condition is a benignproliferative disorder.
 32. The method of claim 31, wherein theproliferative condition is wherein the proliferative condition istuberosclerosis, psoriasis, endometriosis, complex endometrialhyperplasia (cH), atypical endometrial hyperplasia (aH), polyps, orneurofibromatosis.
 33. The method of claim 22, wherein the individual isa human or a non-human mammal.
 34. The method of claim 22, wherein theamount and/or concentration of the ERM family protein is determinedusing a binding agent which binds the ERM family protein.
 35. The methodof claim 34, wherein the binding agent is an antibody, or a functionalfragment thereof. 36-58. (canceled)